Use Of siRNA To Achieve Down Regulation Of An Endogenous Gene In Combination With The Use of A Sense Construct To Achieve Expression Of A Polynucleotide

ABSTRACT

The present invention relates to the combinatorial use of an siRNA targeted against an endogenous gene to knock out or knock down expression of the endogenous gene in a host and a delivery of a polynucleotide encoding the gene in a delivery vehicle/expression vector to the host to provide expression in the host of the protein encoded by the polynucleotide.

FIELD OF THE INVENTION

The present invention relates to the combinatorial use of siRNA targetedagainst a gene to knock down or knock out expression of the endogenousgene, and use of a polynucleotide encoding a exogenous gene to affectexpression of that gene.

BACKGROUND OF THE INVENTION

Relatively recently, researchers observed that double stranded RNA(“dsRNA”) could be used to inhibit protein expression. This ability tosilence a gene has broad potential for treating human diseases, and manyresearchers and commercial entities are currently investing considerableresources in developing therapies based on this technology.

Double stranded RNA induced gene silencing can occur on at least threedifferent levels: (i) transcription inactivation, which refers to RNAguided DNA or histone methylation; (ii) siRNA induced mRNA degradation;and (iii) mRNA induced transcriptional attenuation.

It is generally considered that the major mechanism of RNA inducedsilencing (RNA interference, or RNAi) in mammalian cells is mRNAdegradation. Initial attempts to use RNAi in mammalian cells focused onthe use of long strands of dsRNA. However, these attempts to induce RNAimet with limited success, due in part to the induction of the interferonresponse, which results in a general, as opposed to a target-specific,inhibition of protein synthesis. Thus, long dsRNA is not a viable optionfor RNAi in mammalian systems.

More recently it has been shown that when short (18-30 bp) RNA duplexesare introduced into mammalian cells in culture, sequence-specificinhibition of target mRNA can be realized without inducing an interferonresponse. Certain of these short dsRNAs, referred to as small inhibitoryRNAs (“siRNAs”), can act catalytically at sub-molar concentrations tocleave greater than 95% of the target mRNA in the cell. A description ofthe mechanisms for siRNA activity, as well as some of its applicationsare described in Provost et al. (2002) Ribonuclease Activity and RNABinding of Recombinant Human Dicer, EMBO J. 21(21): 5864-5874; Tabara etal. (2002) The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1and a DexH-box Helicase to Direct RNAi in C. elegans, Cell109(7):861-71; Ketting et al. (2002) Dicer Functions in RNA Interferenceand in Synthesis of Small RNA Involved in Developmental Timing in C.elegans; Martinez et al., Single-Stranded Antisense siRNAs Guide TargetRNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & Zamore (2002) AmicroRNA in a multiple-turnover RNAi enzyme complex, Science 297:2056.

From a mechanistic perspective, introduction of long double stranded RNAinto plants and invertebrate cells is broken down into siRNA by a TypeIII endonuclease known as Dicer. Sharp, RNA interference—2001, GenesDev. 2001, 15:485. Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs. Bernstein, Caudy, Hammond, & Hannon (2001) Rolefor a bidentate ribonuclease in the initiation step of RNA interference,Nature 409:363. The siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition. Nykanen, Haley, & Zamore (2001) ATP requirements and smallinterfering RNA structure in the RNA interference pathway, Cell 107:309.Upon binding to the appropriate target mRNA, one or more endonucleaseswithin the RISC cleaves the target to induce silencing. Elbashir,Lendeckel, & Tuschl (2001) RNA interference is mediated by 21- and22-nucleotide RNAs, Genes Dev. 15:188, FIG. 1.

The interference effect can be long lasting and may be detectable aftermany cell divisions. Moreover, RNAi exhibits sequence specificity.Kisielow, M. et al. (2002) Isoform-specific knockdown and expression ofadaptor protein ShcA using small interfering RNA, J. Biochem. 363:1-5.Thus, the RNAi machinery can specifically knock down one type oftranscript, while not affecting closely related mRNA. These propertiesmake siRNA a potentially valuable tool for inhibiting gene expressionand studying gene function and drug target validation. Moreover, siRNAsare potentially useful as therapeutic agents against: (1) diseases thatare caused by over-expression or misexpression of genes; and (2)diseases brought about by expression of genes that contain mutations.

Successful siRNA-dependent gene silencing depends on a number offactors. One of the most contentious issues in RNAi is the question ofthe necessity of siRNA design, i.e., considering the sequence of thesiRNA used. Early work in C. elegans and plants circumvented the issueof design by introducing long dsRNA (see, for instance, Fire, A. et al.(1998) Nature 391:806-811). In this primitive organism, long dsRNAmolecules are cleaved into siRNA by Dicer, thus generating a diversepopulation of duplexes that can potentially cover the entire transcript.While some fraction of these molecules are non-functional (i.e., inducelittle or no silencing) one or more have the potential to be highlyfunctional, thereby silencing the gene of interest and alleviating theneed for siRNA design. Unfortunately, due to the interferon response,this same approach is unavailable for mammalian systems. While thiseffect can be circumvented by bypassing the Dicer cleavage step anddirectly introducing siRNA, this tactic carries with it the risk thatthe chosen siRNA sequence may be non-functional or semi-functional.

A number of researches have expressed the view that siRNA design is nota crucial element of RNAi. On the other hand, others in the field havebegun to explore the possibility that RNAi can be made more efficient bypaying attention to the design of the siRNA.

To treat various diseases or disorders, the upregulation of certainproteins is desirable but this may not be all that is needed. Forexample, the combinatorial use of siRNA to knock down or knock outexpression of an endogenous protein or a different protein may beneeded. The present invention fulfills this need and provides methods oftreating cancer, especially multiple myeloma.

Cancer, including multiple myeloma are diseases which would benefit fromthe ability to induce apoptosis. Conventional therapies for of multiplemyeloma include chemotherapy, stem cell transplantation, high-dosechemotherapy with stem cell transplantation, and salvage therapy.Chemotherapies include treatment with Thalomid® (thalidomide),bortezomib, Aredia® (pamidronate), steroids, and Zometa® (zoledronicacid). However many chemotherapy drugs are toxic to actively dividingnon-cancerous cells, such as of the bone marrow, the lining of thestomach and intestines, and the hair follicles. Therefore, chemotherapymay result in a decrease in blood cell counts, nausea, vomiting,diarrhea, and loss of hair.

Conventional chemotherapy, or standard-dose chemotherapy, is typicallythe primary or initial treatment for patients with of multiple myeloma.Patients also may receive chemotherapy in preparation for high-dosechemotherapy and stem cell transplant. Induction therapy (conventionalchemotherapy prior to a stem cell transplant) can be used to reduce thetumor burden prior to transplant. Certain chemotherapy drugs are moresuitable for induction therapy than others, because they are less toxicto bone marrow cells and result in a greater yield of stem cells fromthe bone marrow. Examples of chemotherapy drugs suitable for inductiontherapy include dexamethasone, thalidomide/dexamethasone, VAD(vincristine, Adriamycin® (doxorubicin), and dexamethasone incombination), and DVd (pegylated liposomal doxorubicin (Doxil®,Caelyx®), vincristine, and reduced schedule dexamethasone incombination).

The standard treatment for of multiple myeloma is melphalan incombination with prednisone (a corticosteroid drug), achieving aresponse rate of 50%. Unfortunately, melphalan is an alkylating agentand is less suitable for induction therapy. Corticosteroids (especiallydexamethasone) are sometimes used alone for multiple myeloma therapy,especially in older patients and those who cannot tolerate chemotherapy.Dexamethasone is also used in induction therapy, alone or in combinationwith other agents. VAD is the most commonly used induction therapy, butDVd has recently been shown to be effective in induction therapy.Bortezomib has been approved recently for the treatment of multiplemyeloma, but it is very toxic. However, none of the existing therapiesoffer a significant potential for a cure. Thus, there still remains aneed to find a suitable treatment for cancer and multiple myeloma. Thepresent invention fulfills this need.

SUMMARY OF INVENTION

The present invention relates to the combinatorial use of an siRNAtargeted against an endogenous gene to knock out or knock downexpression of the endogenous gene in a host and a delivery of apolynucleotide encoding the gene in a delivery vehicle/expression vectorto the host to provide expression in the host of the protein encoded bythe polynucleotide. A polynucleotide encoding a normal (non faulty)protein (or the protein itself) is administered to the host and isexpressed (in the case of the polynucleotide) so that the normal proteincan perform its necessary function. The siRNA is preferably designed totarget a region of the gene so it either knocks down or knocks outendogenous expression of the faulty protein but at the same time willnot effect exogenous expression of the administered polynucleotideencoding the normal protein.

The invention provides a composition comprising a complex of an eIF5A1siRNA targeted against the 3′ end of eIF5A1, an expression vectorcomprising a polynucleotide encoding a mutant eIF5AI wherein the mutanteIF5A1 is unable to be hypusinated, and wherein the siRNA and theexpression vector are complexed to polyethylenimine to form a complex.

The invention provides a composition comprising an siRNA targetedagainst a target gene to suppress endogenous expression of the targetgene is a subject, and a polynucleotide encoding a target proteincapable of being expressed in the subject. In certain embodiments thepolynucleotide is in RNAI resistant plasmid (will not be suppressed bythe siRNA). The siRNA and the plasmid are preferably complexed topolyethylenimine to form a complex.

In certain embodiments the siRNA has the sequence shown in FIG. 25 andwherein the polynucleotide encoding the mutant eIF5A1 is eIF5A1^(K50R).The expression vector comprises the a polynucleotide encoding a mutanteIF5A1 and a promoter operably linked to provide expression of thepolynucleotide in a subject. The promoter preferably is either tissuespecific or systemic. For example, if the composition is used to treatcancer, then preferably the promoter is tissue specific for the tissuein which the cancer resides. For example, for treating multiple myeloma,it is preferable to use a B cell specific promoter, such as B29. Incertain embodiments, the expression vector comprises a pCpG plasmid.

In certain embodiments, the eIF5A1 siRNA and the expression vectorcomprising the mutant eIF5A1 polynucleotide are independently complexedto polyethylenimine, such as in vivo JetPEI™. In other embodiments, theeIF5A1 siRNA and the expression vector comprising the mutant eIF5A1polynucleotide are complexed together to polyethylenimine.

The present invention further provides a composition comprising aneIF5A1 siRNA targeted against the 3′ end of eIF5A1 and an expressionvector comprising a polynucleotide encoding a mutant eIF5A1 wherein themutant eIF5A1 is unable to be hypusinated, and wherein the siRNA and theexpression vector are delivered to a subject to treat cancer. The cancermay be any cancer including multiple myeloma.

The present invention further provides a method of treating cancercomprising administering composition of the present invention to asubject (including but not limited to mammals and humans).

The composition may be adminstered any acceptable route, such as, butnot limited to intravenously, intra peritoneally, subcutaneously orintra tumorally. The siRNA and the expression vector may be administeredat different times and via different routes or may be administeredtogether at the same time and via the same route. For example, but notlimited to, the siRNA may be delivered naked or complexed to a carriersuch as in vivo jetPEI via IV and the expression vector may beadministered intra tumorally, or both the siRNA and the expressionvector may be administered IV or intratumorally, etc.

The present invention provides a method of inhibiting cancer cell growthand/or killing cancer cells. The present invention also provides amethod of inhibiting or slowing down the ability of a cancer cell tometastasize. Inhibiting cancer growth includes a reduction in the sizeof a tumor, a decrease in the growth of the tumor, and can alsoencompass a complete remission of the tumor. The cancer can be anycancer or tumor, including but not limited to colon cancer, colorectaladenocarcinoma, bladder carcinoma, cervical adenocarcinoma, and lungcarcinoma. Preferably the cancer is multiple myeloma.

In preferred embodiments, the eIF-5A is a mutant that is unable to behypusinated. Exemplary mutants are described herein.

In addition to providing eIF-5A or a polynucleotide encoding eIF-5A to asubject (to provide expression of the eIF-5A), siRNA is provided toknock out or knock down endogenous expression of eIF-5A.

The present invention also provides the use of eIF5A, polynucleotidesencoding eIF5A1 and siRNA against eIF5A1 to make a medicament to treatacner kill multiple myeloma cells in a subject having multiple myeloma.Preferably the polynucleotides encoding a mutant eIF-5A are unable to behypusinated.

The present invention also provides a method of treating sickle cellanemia. A polynucleotide encoding a healthy hemoglobin gene (such asHBB) is administered to a patient suffering from sickle cell anemia. Inconjunction, the patient is also administered siRNA that targets thegene encoding the faulty hemoglobin gene (such as the gene encoding themutant HbS) to knock down or knock out expression of the faulty protein.

The treatment may further comprise administration of other knownmedicines or treatments commonly used in treating sickle cell anemia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the amino acid sequence of human eIF-5A1 and showsvarious important sites.

FIGS. 2A and 2B show that mutation of eIF-5A1 at K50 and K67 increasesaccumulation of transfected protein. See example 1.

FIG. 3 shows that mutation of eIF5A1 at K47, K50 and K67 increasesaccumulation of transfected protein. See example 2.

FIG. 4 shows that mutation of eIF5A1 at K50 and K67 results in inductionof apoptosis when transfected into KAS cells. See example 3.

FIG. 5 shows that mutation of eIF5A1 at K50 and K67 results in inductionof apoptosis when transfected into KAS cells. See example 4.

FIG. 6 shows that mutation of eIF5A1 at K50 and K67 results in inductionof apoptosis when transfected into KAS cells. See example 5.

FIG. 7A shows transfection with siRNA and treating with an adenovirusthat is modified to express eIF-5A1 results in apoptosis in KAS cells.See example 6A. FIG. 7B shows that pre-treatment with eIF5A1 siRNA(against target #1 (SEQ ID NO:1)) the sequence of the siRNA constructshown in FIG. 25 reduced expression of endogenous eIF5A1 but allowsaccumulation of RNAi-resistant eIF5A1^(k50A) expressed by adenovirus.See example 6B. FIG. 7C shows that pre-treatment with eIF5A1 siRNAagainst target #1 prior to adenovirus infection reduces expression ofphosphorylated NF-κB in human multiple myeloma cells. See example 6C.FIG. 7D shows that pre-treatment with eIF5A1 siRNA against target #1prior to Adenovirus infection reduces expression of phosphorylated NF-kBand ICAM-1 in human multiple myeloma cells. See example 6D. FIG. 7Eshows that siRNA-mediated suppression of eIF5A in human multiple myelomacells inhibits LPS-mediated induction of NFkB DNA-Binding Activity. Theinhibition of NFkB activity by eIF5A siRNA could explain it's ability toincrease apoptosis induction when combined with over-expression ofeIF5A^(K50R) since NF-kB regulates many pro-survival and anti-apoptosispathways. FIG. 7F shows that pretreatment of KAS cells with siRNAincreases apoptosis by eIF5A1^(k50R) gene delivery in the presence ofIL-6. See example 6E.

FIG. 8 shows that co-administration of eIF5A1 plasmid and eIF5A1 siRNAdelays growth of multiple myeloma subcutaneous tumours. The data shownis the tumor volume for all the mice in each group. See example 7.

FIG. 9 shows that co-administration of eIF5A1 plasmid and eIF5A1 siRNAdelays growth of multiple myeloma subcutaneous tumours. The data shownis the average tumor volume per group +/−standard error. See example 7.

FIG. 10 shows that co-administration of eIF5A1 plasmid and eIF5A1 siRNAreduces weight of multiple myeloma subcutaneous tumours. See example 7.

FIG. 11 shows that co-administration of eIF5A1 plasmid and eIF5A1 siRNAdelays growth of multiple myeloma subcutaneous tumours and results intumour shrinkage. See Example 8.

FIG. 12 shows that administration of eIF5A1 siRNA intra-venously (i.v.)and PEI/eIF5A1K50R plasmid complexes intra-tumourally (i.t.) results intumour shrinkage of multiple myeloma subcutaneous tumours. See example9.

FIG. 13A shows that treatment with eIF5A1 plasmid and eIF5A1 siRNAdelays growth of multiple myeloma subcutaneous tumours and results intumour shrinkage. FIG. 13B shows that co-administration of eIF5A1plasmid and eIF5A1 siRNA results in tumour shrinkage. FIG. 13C showsthat administration of eIF5A1 siRNA intra-venously (i.v.) andPEI/eIF5A1^(K50R) plasmid complexes intra-tumourally (i.t.) results intumour shrinkage of multiple myeloma subcutaneous tumours.

FIG. 14 shows that intra-venous co-administration of eIF5A1 plasmid andeIF5A1 siRNA delays growth of multiple myeloma subcutaneous tumours. Seeexample 10.

FIG. 15 shows that administration of eIF5A1 siRNA intra-venously (i.v.)and 5 PEI/eIF5A1K5OR plasmid complexes intra-venously (i.v.) orintra-peritoneal (i.p.) delays growth of multiple myeloma subcutaneoustumours. See example 11.

FIG. 16 shows that treatment with eIF5A1 plasmid and eIF5A1 siRNA delaysgrowth of multiple myeloma subcutaneous tumours.

FIGS. 17A and 17B show that co-administration of eIF5A1 plasmid andeIF5A1 siRNA delays growth of multiple myeloma subcutaneous tumours andresults in tumour shrinkage. See example 12.

FIGS. 18A and 18B show that administration of eIF5A1 siRNAintra-venously (i.v.) and PEI/eIF5A1K5OR plasmid complexesintra-tumourally (i.t.) results in tumour shrinkage of multiple myelomasubcutaneous tumours. See example 13.

FIG. 19 shows co-administration of eIF5A1^(K50R) plasmid, driven byeither the EF1 or B29 promoter, and eIF5A1 siRNA delays growth ofmultiple myeloma subcutaneous tumours and results in tumour shrinkage(KAS-SQ-5). See example 14.

FIG. 20 shows co-administration of eIF5A1 siRNA increases anti-tumoreffect of eIF5A1^(K50R) plasmid, driven by either the EF1 or B29promoter, on multiple myeloma subcutaneous tumours and results inreduced tumor burden (KAS-SQ-5). See example 15.

FIG. 21 shows eIF5A1 siRNA synergistically increases apoptosis resultingfrom infection with Ad-eIF5A in lung adenocarcinoma cells. See example16.

FIG. 22 shows the map of pExp5A, the construction of which is describedin Example 17.

FIGS. 23A and 23 B show the predicted sequence of pExp5A (3371 bp). Seeexample 17.

FIGS. 24A and 24B show the rexpression of eIF5A^(K50R) in various celllines. See example 18.

FIG. 25 shows the target sequence and the sequence of a preferred eIF5A1siRNA.

FIG. 26 provides the results of efficacy studies in multiple myeloma.See example 21.

FIG. 27 provides the results of efficacy studies in multiple myeloma.See example 21.

FIG. 28 provides the sequence of eIF5A1^(K50R) cDNA.

FIG. 29 provides the alignment of human eIF-5A against humaneIF5A^(K50R).

FIG. 30 shows the effect of DNA:siRNA ratio on HA-eIF5A1^(K50R)expression. See example 23.

FIG. 31 shows the effect of DNA:siRNA ratio on apoptosis induced bynanoparticle transfection. See example 24.

FIG. 32 shows administration of PEI complexes (N/P=6 or 8) containingeIF5A1K5OR plasmid and eIF5A1 siRNA (siSTABLE or non-siSTABLE) inhibitsgrowth of multiple myeloma subcutaneous tumours and results in tumourshrinkage. See example 25.

FIG. 33 shows that the JET PEI™ nanoparticles are being effectivelytaken up by tumour tissue and that nanoparticles are delivering plasmidand siRNA to the same cell. See example 26.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the combinatorial use of an siRNAtargeted against an endogenous gene to knock out or knock downexpression of the endogenous gene in a subject and a of a polynucleotideencoding the gene in a delivery vehicle/expression vector provided tothe subject to provide expression in the host of the protein encoded bythe polynucleotide.

This combination is useful in treating a subject with a disease orcondition caused by the existence of a faulty or mutant protein, i.e.where the protein produced in the subject is unable to perform itnecessary function or alternatively, fowls up a metabolic pathway orbiomolecule interaction because of its faulty structure. The siRNA isdesigned to target the gene encoding the faulty protein, and knock downor knock out expression of that faulty protein. A polynucleotideencoding a normal (non faulty) protein is administered to the subjectand is expressed in the subject so that the normal protein is availableto perform its necessary function.

In another embodiment, instead of administering the polynucleotideencoding the desired protein, the protein is administered to thesubject. The terms protein, peptide and polypeptide are used hereininterchangeably.

The siRNA is preferably designed to target a certain region of the geneso it either knocks down or knocks out endogenous expression of thefaulty protein but at the same time will not effect exogenous expressionof the administered polynucleotide encoding the normal protein. Forexample, the siRNA may target the 3′UTR so it does not effect exogenousexpression of the administered sense construct (the polynucleotideencoding the protein). By knocking down or knocking out endogenousexpression of the faulty gene, there will be less or none of the faultyprotein to compete with the normal protein expressed from the exogenouspolynuclecotide.

One example of a disease state where this application would be usefulconcerns sickle cell anemia. Sickle cell anemia is a blood disorder thataffects hemoglobin, the protein found in red blood cells (RBCs) thathelps carry oxygen throughout the body.

Sickle cell anemia occurs when a person inherits two abnormal genes (onefrom each parent) that results in expression of a mutant hemoglobin(Hbs). The mutant hemoglobin causes the RBCs to change shape. Red bloodcells with normal hemoglobin (hemoglobin A, or HbA) move easily throughthe bloodstream, delivering oxygen to all of the cells of the body. Theycan easily “squeeze” through even very small blood vessels. Sickle cellanemia occurs because the abnormal form of hemoglobin (HbS) tends toclump together, making red blood cells sticky, stiff, and more fragile,and causing them to form into a curved, sickle shape.

Although several hundred HBB gene variants are known, sickle cell anemiais most commonly caused by the hemoglobin variant HbS. In this variant,the hydrophobic amino acid valine takes the place of hydrophilicglutamic acid at the sixth amino acid position of the HBB polypeptidechain. This substitution creates a hydrophobic spot on the outside ofthe protein structure that sticks to the hydrophobic region of anadjacent hemoglobin molecule's beta chain. This clumping together(polymerization) of HbS molecules into rigid fibers causes the“sickling” of red blood cells.

Polymerization occurs only after red blood cells have released theoxygen molecules that they carry to various tissues throughout the body.Once red blood cells return to the lungs where hemoglobin can bindoxygen, the long fibers of HbS molecules depolymerize or break apartinto single molecules. Cycling between polymerization anddepolymerization causes red blood cell membranes to become rigid. Therigidity of these red blood cells and their distorted shape when theyare not carrying oxygen can result in blockage of small blood vessels.This blockage can cause episodes of pain and can damage organs.

Sickle cell anemia is an autosomal recessive genetic disorder. For thedisease to be expressed, a person must inherit either two copies of HbSvariant or one copy of HbS and one copy of another variant. Carriers,who have one copy of the normal HBB gene (HbA) and one copy of HbS, aredescribed as having sickle cell trait and do not express diseasesymptoms.

Thus, one embodiment of the present invention provides a method oftreating subjects with sickle cell anemia. siRNA targeted to the HBBgene is administered to the patient. The siRNA is designed to knock downand preferably knock out the expression of the Hbs variant ofhemoglobin. A polynucleotide encoding a normal hemoglobin is provided tothe subject so the subject expresses a normal hemoglobin. The siRNA isalso designed so that it will not interfere with expression of theexogenous polynucleotides encoding the normal hemoglobin. Thus, thesubject no longer makes the variant hemoglobin (or makes substantiallyless) and instead makes normal healthy hemoglobin, resulting in morenormal red blood cells, which function normally.

The present invention is also useful in situations where a posttranslational modification occurs to the protein, which causes or leadsto a disease state. siRNA is used to knock down expression of theendogenous protein so none or less is available for the posttranslational modification. Then, a polynucleotide encoding a protein isprovided to the patient for exogenous expression. The protein ismodified so that it is unable to be post translationally modified. Thisprotein is then available to the body for its appropriate use, but willnot lead to the disease state because it is not able to be posttranslationally modified. One skilled in the art would understanddifferent post translational modifications. For example, aftertranslation, the posttranslational modification of amino acids extendsthe range of functions of the protein by attaching to it otherbiochemical functional groups such as acetate, phosphate, various lipidsand carbohydrates, by changing the chemical nature of an amino acid(e.g. citrullination) or by making structural changes, like theformation of disulfide bridges. Also, enzymes may remove amino acidsfrom the amino end of the protein, or cut the peptide chain in themiddle. For instance, the peptide hormone insulin is cut twice afterdisulfide bonds are formed, and a propeptide is removed from the middleof the chain; the resulting protein consists of two polypeptide chainsconnected by disulfide bonds. Also, most nascent polypeptides start withthe amino acid methionine because the “start” codon on mRNA also codesfor this amino acid. This amino acid is usually taken off duringpost-translational modification. Other modifications, likephosphorylation, are part of common mechanisms for controlling thebehavior of a protein, for instance activating or inactivating anenzyme. Another post translational modification includes thehypusination of eukarotic initiation factor 5A (eIF5A) by deoxyhypusinesynthase (DHS).

Thus, the invention provides a method of altering expression of a genein a subject, wherein a polynucleotide encoding a protein is provided toa patient and is expressed in the patient. The protein may be anormal/wild type protein or a mutated protein. Expression of thecorresponding endogenous gene is suppressed with the siRNA that isadministered to the subject.

The method further comprises providing a construct comprising apolynucleotide encoding the target protein wherein the polynucleotide isexpressed in the subject to produce the target protein. In certainembodiments where the endogenous gene expresses a faulty protein, thepolynucleotide is designed to encode either a normal/healthy protein.The siRNA is administered to suppress expression of the faultyendogenous protein. In certain embodiments where the endogenous geneexpresses a normal healthy protein, the polynucleotide is designed toencode a mutant protein that can not be postranslationally modified aswould occur with a normal/healthy or non mutant protein. The siRNA isadministered to suppress expression of the endogenous protein so thereis less of this protein to be available for posttranslationalmodification.

In certain embodiments, the siRNA is chosen or designed to targetregions of the gene so as to not effect expression of the exogenouspolynucleotide. For example, the siRNA may target the 3′ UTR or 3′ end.

The siRNA may be delivered to the patient as either naked siRNA or nakedsiRNA stabilized for serum. The siRNA may by either injectedsystemically, i.e. IP or IV. Alternatively, the siRNA may be injected ordelivered locally to the desired area of the body. In certainembodiments, the siRNA may be administered in a delivery vehicle such asbut not limited to dendrimers, liposomes, or polymers.

The polynucleotides encoding the desired protein may be administeredthrough any delivery means that provide or allow expression of thenucleotide. The term polynucleotide and nucleotide are used hereininterchangeably. Delivery may be through any viral or non-viralmechanism, such as but not limited to plasmids, expression vectors,viral constructs, adenovirus constructs, dendrimers, liposomes, orpolymers.

In certain embodiments an expression plasmid having reduced CpGdinucleotides is used to express the polynucleotides. Any promotercapable of promoting expression of the polynucleotide may be used, whichmay be chosen based on the application desired for the therapy. Forexample, for killing multiple myeloma, a promoter specific for B cellsmay be desirable, such as human B29 promoter/enhancer. In otherembodiments, the promoter may be another tissue specific promoter, ormay be a system promoter.

The polynucleotides encoding the target protein may be delivered throughIV or subcutaneous injection or any other biologically suitable deliverymechanism.

Alternatively, the polynucleotides may be delivered in liposomes or anyother suitable “carrier” or “vehicle” that provides for delivery of theDNA (or plasmid or expression vector) to the target tumor or cancercells. See for example, Luo, Dan, et al., Nature Biotechnology, Vol. 18,January 2000, pp. 33-37 for a review of synthetic DNA delivery systems.Thus, it may be preferable to deliver the nucleotides/plasmid/expressionvector via a vehicle of nanometer size such as liposomes, dendrimers ora similar non-toxic nano-particle. The vehicle preferably protects thenucleotides/plasmid/expression vector from premature clearance or fromcausing an immune response while delivering an effective amount of thenucleotides/plasmid/expression vector to the subject, tumor or cancercells. Exemplary vehicles may range from a simple nano-particleassociated with the nucleotides/plasmid/expression vector to a morecomplex pegylated vehicle such as a pegylated liposome having a ligandattached to its surface to target a specific cell receptor.

Liposomes and pegylated liposomes are known in the art. In conventionalliposomes, the molecules to be delivered (i.e. small drugs, proteins,nucleotides or plasmids) are contained within the central cavity of theliposome. One skilled in the art would appreciate that there are also“stealth,” targeted, and cationic liposomes useful for moleculedelivery. See for example, Hortobagyi, Gabriel N., et al., J. ClinicalOncology, Vol. 19, Issue 14 (July) 2001:3422-3433 and Yu, Wei, et al.,Nucleic Acids Research. 2004, 32(5);e48. Liposomes can be injectedintravenously and can be modified to render their surface morehydrophilic (by adding polyethylene glycol (“pegylated”) to the bilayer,which increases their circulation time in the bloodstream. These areknown as “stealth” liposomes and are especially useful as carriers forhydrophilic (water soluble) anticancer drugs such as doxorubicin andmitoxantrone. To further the specific binding properties of a drugcarrying liposome to a target cell, such as a tumor cell, specificmolecules such as antibodies, proteins, peptides, etc. may be attachedon the liposome surface. For example, antibodies to receptors present oncancer cells maybe used to target the liposome to the cancer cell. Inthe case of targeting multiple myeloma, folate, II-6 or transferrin forexample, may be used to target the liposomes to multiple myeloma cells.

Dendrimers are also known in the art and provide a preferable deliveryvehicle. See for example Marjoros, Istvan, J., et al, “PAMAMDendrimer-Based Multifunctional Conjugate for Cancer Therapy: Synthesis,Characterization, and Functionality,” Biomacromolecules, Vol. 7, No. 2,2006; 572-579, and Majoros, Istvan J., et al., J. Med. Chem, 2005. 48,5892-5899 for a discussion of dendrimers.

In a preferred embodiment, the delivery vehicle comprises apolyethylenimine nanoparticle. An exemplary polyethyleniminenanoparticle is the in vivo-jetPEI™, currently produced by PolyplusTransfection, Inc. In vivo-jetPEI™ is cationic polymer transfectionagent useful as a DNA and siRNA delivery agent. In vivo-jetPEI™ fromPolyplus Transfection is a linear polyethylenimine reagent that providesreliable nucleic acid delivery in animals. It is used for gene therapy(Ohana et al., 2004. Gene Ther Mol Bio 8:181-192; Vernejoul et al.,2002. Cancer Research 62:6124-31), RNA interference, (Urbain-Klein etal., 2004. Gene therapy 23:1-6; Grezelinski et al., 2006. Human GeneTherapy 17:751-66), and genetic vaccination (Garzon et al., 2005.Vaccine 23:1384-92). In vivo JET-PEI is currently in use in humanclinical trials as a delivery vector for cancer gene therapy (Lemkine etal., 2002. Mol. Cell. Neurosci. 19:165-174).

In vivo-jetPEI™ condenses nucleic acids into roughly 50 nmnanoparticles, which are stable for several hours. As a result of thisunique protection mechanism, aggregation of blood cells followinginjection is reduced compared to other reagents thereby preventingrestricted diffusion within a tissue, erythrocyte aggregation andmicroembolia. These nanoparticles are sufficiently small to diffuse intothe tissues and enter the cells by endocytosis. In vivo-jetPEI™ favorsnucleic acids release from the endosome and transfer across of thenuclear membrane.

In a preferred embodiment, both the siRNA and a vector/plasmidcomprising the polynucleotide are administered to the subject via an invivo-jetPEI™ complex. The siRNA and the vector/plasmid comprising thepolynucleotide maybe complexed together via a polymer complex such aspolyethylenimine or the in vivo jetPEI™ complex or may separatelycomplexed to a polymer. For instance, where the siRNA and thevector/plasmid comprising the polynucleotide are to be administeredseparately to the subject (separately in the meaning of time and/ordelivery site) it is preferable to have the siRNA and the polynucleotidecomplexed to a different carrier. Where the administration will be atthe same time and at the same site, it may be preferable to complex thesiRNA and the polynucleotide together.

In another embodiment, instead of a plasmid or vector being administeredto deliver a polynucleotide that will be expressed in the subject, theprotein per se is delivered to the subject. The protein may be eitherisolated or may be synthetic.

One embodiment of the present invention provides a method of treatingcancer in a subject, including mammals and humans. Treating cancerincludes, but is not limited to inducing apoptosis in cancer cells,killing cancer cells, reducing the number of cancer cells and reducingtumor volume/weight. The method comprises administering a compositioncomprising eIF5A1 siRNA and a polynucleotide encoding a mutant eIF5A1.The composition and eIF5A1 siRNA and a polynucleotide encoding a mtuanteIF5A1 are discussed herein below.

All cells produce eukaryotic initiation factor 5A (“eIF-5A”) (or alsoreferred to herein as “factor 5A”). Mammalian cells produce two isoformsof eIF-5A1 (eIF-5A1 and eIF-5A2). eIF-5A1 has been referred to asapoptosis-specific eIF-5A, as it is upregulated in cells undergoingapoptosis. Human eIF-5A1 has the accession number NM 001970 and is shownin FIG. 1. It is believed that eIF-5A1 is responsible for shuttling outof the nucleus subsets of mRNAs encoding proteins necessary forapoptosis. eIF-5A2 has been referred to as proliferation eIF-5A as it isbelieved to be responsible for shuttling out of the nucleus subsets ofmRNAs encoding proteins necessary for cellular proliferation. See Liu &Tartakoff (1997) Supplement to Molecular Biology of the Cell, 8, 426a.Abstract No. 2476, 37th American Society for Cell Biology AnnualMeeting, and Rosorius et al. (1999) J. Cell Science, 112, 2369-2380.

Both factor 5As are post translationally modified by deoxyhypusinesynthase (“DHS”). DHS hypusinates the eIF-5As. Hypusine, a unique aminoacid, is found in all examined eukaryotes and archaebacteria, but not ineubacteria, and eIF-5A is the only known hypusine-containing protein.Park (1988) J. Biol. Chem., 263, 7447-7449; Schumann & Klink (1989)System. Appl. Microbiol., 11, 103-107; Bartig et al. (1990) System.Appl. Microbiol., 13, 112-116; Gordon et al. (1987a) J. Biol. Chem.,262, 16585-16589. Hypusinated eIF-5A is formed in two post-translationalsteps: the first step is the formation of a deoxyhypusine residue by thetransfer of the 4-aminobutyl moiety of spermidine to the α-amino groupof a specific lysine of the precursor eIF-5A catalyzed by deoxyhypusinesynthase. The second step involves the hydroxylation of this4-aminobutyl moiety by deoxyhypusine hydroxylase to form hypusine.

The amino acid sequence of eIF-5A is well conserved between species, andthere is strict conservation of the amino acid sequence surrounding thehypusine residue in eIF-5A, which suggests that this modification may beimportant for survival. Park et al. (1993) Biofactors, 4, 95-104. Thisassumption is further supported by the observation that inactivation ofboth isoforms of eIF-5A found to date in yeast, or inactivation of theDHS gene, which catalyzes the first step in their activation, blockscell division. Schnier et al. (1991) Mol. Cell. Biol., 11, 3105-3114;Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J.Biol. Chem., 273, 1677-1683. However, depletion of eIF-5A protein inyeast resulted in only a small decrease in total protein synthesissuggesting that eIF-5A may be required for the translation of specificsubsets of mRNA's rather than for protein global synthesis. Kang et al.(1993), “Effect of initiation factor eIF-5A depletion on cellproliferation and protein synthesis,” in Tuite, M. (ed.), ProteinSynthesis and Targeting in Yeast, NATO Series H. The recent finding thatligands binding eIF-5A share highly conserved motifs also supports theimportance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276, 2555-2561.In addition, the hypusine residue of modified eIF-5A was found to beessential for sequence-specific binding to RNA, and binding did notprovide protection from ribonucleases.

The present inventors have shown that when polynucleotides encodingeIF-5A are administered to cells, there is an increase in apoptosisthose cells. They have shown that they have been able to push cancercells into apoptosis by administering eIF-5Al polynucleotides that arethen expressed in the cancer cells. See co-pending application Ser. Nos.10/200,148; 11/287,460; 11/293,391 and 11/637,835, all of which areincorporated by reference in their entireties.

The present inventors have additionally determined that when cells havea build up of the hypusinated form of factor 5A, the cells enter into asurvival mode and do not undergo apoptosis as they normally would overtime. Notably, in cancer cells, there is a significant amount ofhypusinated factor 5A and thus, the cells do not enter into apoptosis(and do not die). Thus, to treat cancer by killing the cancer cells(push the cancer cells to enter into the apoptosis pathway), apolynucleotide encoding eIF-5A1 is administered to the subject or to thecancer cells or tumor to provide increased expression of eIF-5A1, whichin turn causes apoptosis in the cancer cells and ultimately cell deathand tumor shrinkage. However, if one were to only providepolynucleotides encoding the eIF-5A1 protein to up regulate geneexpression of eIF-5Al and not also use siRNA to knock down endogenousexpression of eIF-5A1, there is a tug of war: the eIF-5A1 expressiondirecting the cells towards the apoptosis pathway competes with thepresence of the hypusinated factor 5A directing the cells towards thecell survival pathway. The present invention eliminates this tug of warand represents an improvement over only increasing expression ofeIF-5A1. The polynucleotides administered to the subject or cell aremutated so that the resulting expressed protein can not be hypusinated.In addition endogenous expression of factor 5A is knocked out/down withsiRNA targeted against eIF-5A so there is none/less endogenous eIF-5A1around to by hypusinated. Thus, since there is no (or substantiallyless) hypusinated eIF-5A in the cells, they are not pushed into survivalmode.

The polynucleotide encoding a mutated eIF-5A1 is preferably mutated sothat it can not be hypusinated and thus will not be available to drivethe cell into survival mode. For example, in one embodiment, thepolynucleotide encoding eIF-5A is mutated to so that the lysine (K) atposition 50, which is normally hypusinated by DHS, is changed to analanine (A) (which can not be hypusinated). This mutant is denoted asK50A.

In another embodiment, the lysine at position 67 is changed to anarginine (R). This mutant is denoted as (K67R). In another embodimentthe lysine (K) at position 67 is changed to an alanine (A) and isdenoted as (K67A). In another embodiment, the lysine (K) at position 50is changed to an arginine (K50R) and another embodiment provides amutant where the lysine (K) at position 47 is changed to an arginine(K47R).

In other embodiments, a double mutant is used. One double mutant iswhere the lysine (K) at position 50 is changed to an arginine (R) andthe lysine (K) at position 67 is changed to a arginine (R). This doublemutant is referred to as K50R/K67R. This double mutant is similarlyunable to be hypusinated but the changes in the amino acids do not alterthe 3-D structure of eIF-5A1 as much as the single mutation (K50A). Thedouble mutation thus provides a protein that is very similar in 3-Dshape and folding as the wild type and thus is more stable than thesingle mutant. Being more stable, it exists longer in the body toprovide longer therapeutic benefit. Thus, the body will have the factor5A it needs for normal cell function but it will not be able tohypusinated so the cells do not get locked into the cell survival modeand escape apoptosis.

Another double mutant is where the lysine (K) at position 47 is changedto an arginine (R) and the lysine at position 50 is changed to anarginine (R). This mutant is denoted as (K47R/K50R). The inventionprovides another double mutant where the lysine (L) at position 50 ischanges to an alanine (A) and the lysine at position 67 is changes to analanine (A). This mutant is denoted as (K50A/K67A).

Because the body needs factor 5A for normal cell survival and healthycell proliferation, it is preferable not to shut off expressioncompletely in the subject with the siRNA, if the siRNA is deliveredsystemically. Control of eIF-5A expression can be achieved by eitherusing an siRNA that is not as good at shutting off expression (i.e.shuts down or reduces expression but does not completely shut offexpression) or alternatively, or utilizing a dosing and/or treatmentregimen to balance expression levels to allow normal growth andfunctioning of healthy cells but also to push cancerous cells toapoptosis.

Alternatively, one may utilize local delivery of siRNA. If the siRNA isdelivered locally to the cancer cell or tumor, then the expression ispreferably knocked out. By knocking out expression, there is no factor5A around that can be hypusinated and thus there is no hypusinatedeIF-5A to lock the cells into survival mode. Since the siRNA isdelivered locally to the cancer or tumor, there is no need to haveeIF-5A available for regular cell growth.

In certain embodiments, the endogenous gene is eIF5A1. siRNA targetedagainst eIF5A1 is administered to the subject to suppress expression ofthe endogenous eIF-5A1. In certain embodiments the siRNA comprises SEQID NO:1 or SEQ ID NO:2 or is any siRNA targeted against eIF5A1 that willsuppress expression of endogenous eIF-5A1. In certain embodiments, theeIF5A1 is human eIF-5A1 (shown in FIG. 1) and the subject is a human.Other siRNAs targeted against human eIF-5A1 are known and disclosed inco-pending application Ser. Nos. 11/134,445; 11/287,460; 11/184,982;11/293,391; 11/725,520; 11/725,470; 11/637,835. In other embodiments,the subject is a mammal and the eIF5A1 is specific to the mammal. Forexample, the subject is a dog and the eIF5A1 is canine eIF5A1. Incertain embodiments, the siRNA consists essentially of the siRNAconstruct shown in FIG. 25. For example, the siRNA contains nucleicacids targeted against the eIF5A1 but also contains overhangs such as Uor T nucleic acids or also contains tags, such as a his tag (oftenreferred to as HA tag which is often used in in vitro studies).Molecules or additional nucleic acids attached at either the 5′ or 3′end (or even within the consecutive string of nucleic acids shown inFIG. 25, for example) may be included and fall within the “consistingessentially of” as long as the siRNA construct is able to reduceexpression of the target gene. Preferably the siRNA targets regions ofthe eIF5A1 gene so as to not effect expression of the exogenouspolynucleotide. For example the eIF5A1 siRNA targets the 3′ UTR or the3′ end. The siRNA shown in FIG. 25 an exemplary eIF5A1 siRNA.

The polynucleotide encodes eIF5A1 wherein the polynucleotide is mutatedto encode an eIF5A1 variant. The mutated eIF5A1 is designed so that thevariant eIF5A1 can not be post translationally modified (can not behypusinated). Exemplary mutants are discussed herein above.

In the case of cancer involving solid tumors, it may be desirable todeliver the siRNA directly to the tumor. The siRNA maybe administeredseparately with respect to time as well as the delivery site from thepolynucleotide or may administered together at the same time and/or atthe same delivery site. One skilled in the art would understand that thetiming of administration of the siRNA may be necessarily administeredwhen the endogenous protein is being translated and not after it isalready made.

Although the present inventors have earlier shown that eIF5A1 is nontoxic to normal tissue (see pending application Ser. No. 11/293,391,filed Nov. 28, 2005, which is incorporated herein by reference in itsentirety), a delivery complex (as compared to direct administration ofthe eIF5A polynucleotides/plasmid/expression vector) may be preferred. Apreferred delivery system provides an effective amount of eIF5A1 to thesubject, tumor or group of cancer cells, as well as preferably providesa targeted delivery to the tumor or group of cancer cells. Thus, incertain embodiments, it is preferable to deliver the eIF5A1nucleotides/plasmid/expression vector via a vehicle of nanometer sizesuch as liposomes, dendrimers or a similar non-toxic nano-particle suchas a polyethylenimine polymer (such as an in vivo JetPEI™ complex).

The eIF5A1 protein may also be delivered directly to the site of thetumor. One skilled in the art would be able to determine the dose andlength of treatment regimen for delivery of eIF5A1 protein.

The molecular basis for the induction of apoptosis by eIF5A1 isdiscussed below.

Death Receptor Signaling

Treatment of cancer cells with Ad-eIF5A1 (adenovirus with a wild typeeIF5A1) or Ad-eIF5A1(K50A) induces activation of caspase 8, which isinitiated by death receptor-ligand binding, and caspase 3, theexecutioner caspase. These are likely to be indirect effects of eIF5A1,and the fact that caspase 8 and caspase 3 are also activated followingtreatment with eIF5A1(K50A), which cannot be hypusinated, indicates thatthe effect is attributable to lysineso eIF5A1. Treatment with Ad-eIF5A1also appears to result in up-regulation of death receptors as shownpreviously with upregulation of TNFR1.

Mitochondrial Pathway

Direct or indirect involvement of lysine₅₀ eIF5A1 in the mitochondrialpathway for apoptosis is supported by a number of observations includingthe finding that caspase 9 is activated by treatment of cancer cellswith either eIF5A1 or eIF5A1(K50A). As well, p53, which plays a role inactivation of the mitochondrial apoptotic pathway, appears to beregulated by eIF5A1. For example, treatment of cancer cells withActinomycin D up-regulates p53, and this up-regulation of p53 isinhibited by eIF5A1 siRNA. Consistent with this, treatment of cancercells with Ad-eIF5A1 up-regulates p53 mRNA. Treatment of cancer cellswith eIF5AI also induces migration of Bax from the cytosol tomitochondria, ensuing loss of mitochondrial membrane potential andrelease of cytochrome C from the intramitochondrial space into thecytosol. In addition, this treatment results in up-regulation of cleavedBcl2, Bim and spliced Bim, which are all pro-apoptotic.

MAPK Signaling

In addition, the present inventors have obtained evidence for theinvolvement of eIF5A1 in MAPK signaling related to apoptosis. Forexample, treatment of cancer cells with Ad-eIF5A1 up-regulated P-JNK,which in turn inhibits anti-apoptotic Bcl2. In addition Ad-eIF5A1 andAd-eIF5A1(K50A) both induce the formation of P-p38, which can in turninitiate apoptosis by impacting a variety of pro-apoptotic agentsincluding TNFR1 & TNF; FAS & FASL; caspase 8; Bid; Cytochrome C andCapase 3.

NF-κB Signaling

There is evidence that NF-κB signaling supports myeloma growth. Forexample, myeloma cell adhesion to bone marrow stromal cells inducesNF-κB-dependent transcriptional up-regulation of IL-6, which is both agrowth and anti-apoptotic factor in multiple myeoloma [Chauhan et al.(1996) Blood 87, 1104.] In addition, TNF-α secreted by myeloma cellsactivates NF-κB in bone marrow stromal cells, thereby up-regulating IL-6transcription and secretion. TNF-α also activates NF-κB in myeloma cellsresulting in up-regulation of the intracellular adhesion molecule-1(ICAM-1; CD54) and the vascular cell adhesion molecule-1 (VCAM-1; CD106)on both myeloma cells and bone marrow stromal cells [Hideshima et al.(2001) Oncogene 20, 4519]. This in turn enhances the association ofmyeloma cells with bone marrow stromal cells [Hideshima et al. (2001)Oncogene 20, 4519]. Conversely, these effects are inhibited by blockingTNFα-induced NF-κB activation [Hideshima et al. (2001) Oncogene20,4519]. Indeed, it seems likely that NF-κB mediates protection againstTNFα-induced apoptosis in myeloma cells [Hideshima et al. (2002) JBC277, 16639]. These and other observations have prompted the view thatNF-kB signaling may be an attractive target for multiple myelomatherapies.

The inventors have shown that eIF5A1 siRNA inhibits both the activationof NF-κB and the formation of ICAM-1 in human myeloma cells. Theseobservations indicate that eIF5A1 plays a role in NF-κB activation, andinasmuch as the ensuing effects of NF-κB activation are pro-survival innature, we predict that this activation is mediated, directly orindirectly, by hypusinated eIF5A1.

IL-1

Over-production of the pro-inflammatory cytokine, IL-1, by myeloma cellsis a characteristic feature of multiple myeloma that leads todeterioration of bone tissue. eIF5A1 siRNA has been shown todramatically reduce the overproduction of IL-1 induced by an LPSchallenge in mice.

One embodiment of the present invention provides a method of treatingmultiple myeloma. Multiple myeloma (“MM”) is a progressive and fataldisease characterized by the expansion of malignant plasma cells in thebone marrow and by the presence of osteolytic lesions. Multiple myelomais an incurable but treatable cancer of the plasma cell. Plasma cellsare an important part of the immune system, producing immunoglobulins(antibodies) that help fight infection and disease. Multiple myeloma ischaracterized by excessive numbers of abnormal plasma cells in the bonemarrow and overproduction of intact monoclonal immunoglobulins (IgG,IgA, IgD, or IgE; “M-proteins”) or Bence-Jones protein (free monoclonallight chains). Hypocalcaemia, anemia, renal damage, increasedsusceptibility to bacterial infection, and impaired production of normalimmunoglobulin are common clinical manifestations of multiple myeloma.Multiple myeloma is often also characterized by diffuse osteoporosis,usually in the pelvis, spine, ribs, and skull.

The present invention seems to be well suited to treat multiple myelomabecause of the stimulation feedback loop found in multiple myeloma. Forinstance, multiple myeloma produces Il-1 in low concentrations in bonemarrow. The Il-1 in turn stimulates stromal cells to produce IL-6, whichthen goes onto stimulate growth of the multiple myeloma. The inventorshave previously shown (see pending application Ser. Nos. 11/725,539 and11/184,982) that siRNA directed against eIF-5A1 was able to inhibitexpression of proinflammatory cytokines, such as Il-1; TNF-α, and Il-8).Thus, the siRNA would not only knock down expression of eIF-5A so lessis available for hypusination, it would also cut off or decrease theIl-1/Il-6 feedback loop.

An siRNA targeting human eIF5A was used to suppress levels of endogenoushypusinated eIF5A in tumours, while an RNAi-resistant plasmid expressinga mutant of eIF5A (eIF5A^(K50R)), that is incapable of beinghypusinated, was used to raise the levels of unmodified eIF5A in vivo.Intra-tumoural injection of PEI nanocomplexes containing eIF5A siRNAinhibited MM tumour growth by more than 80% (***p=0.0003) versuscomplexes containing a control siRNA, indicating that suppressing levelsof hypusinated eIF5A has an anti-tumoural effect. PEI complexescontaining an eIF5A^(K50R) expression plasmid had a similar effect andinhibited tumour growth by more than 70% (**=p 0.001) versus complexescontaining a control plasmid. Thus, MM tumour growth can be inhibitedeither by suppression of the growth-promoting hypusinated eIF5A or byincreasing levels of the pro-apoptotic unhypusinated form of eIF5A.Intra-tumoural delivery of complexes containing both eIF5A siRNA andRNAi-resistant eIF5A^(K50R) plasmid had a synergistic effect on tumourgrowth and resulted in significant tumour shrinkage, inhibiting tumourgrowth by 94% (***p=0.0002). Intra-venous delivery of eIF5AsiRNA/eIF5A^(K50R) PEI complexes also efficiently reduced tumour growthby 95% (**p=0.002) indicating systemic delivery of the therapeutic isfeasible.

Both local and systemic delivery of eIF5A siRNA/eIF5A^(K50R) pDNA PEIcomplexes resulted in a significant anti-tumoural response in multiplemyeloma.

The present invention further provides a composition useful in thetreatment of cancer, including multiple myeloma. In a preferredembodiment, the composition is a complex of a plasmid DNA encodingpoint-mutated eIF5A1 that cannot be hypusinated and eIF5A1 siRNA thatselectively suppresses endogenous human eIF5A1 but has no effect on thepoint-mutated eIF5A1 encoded by the plasmid. eIF5A siRNAs andpolynucleotides encoding mutant eIF5A are discussed above. The plasmidDNA and the siRNA are both preferably complexed to PEI(polyethylenimine) nanoparticles. They may be complexed separately andadministered separately or together or they may be complexed together.The DNA and the RNA bind to positively charged amino groups on the PEIand are released when the nanoparticles are taken up into cells. It hasbeen demonstrated that PEI-nucleic acid complexes are effectively takenup into both dividing and non-dividing cells.

The plasmid DNA preferably encodes eIF5AI(K50R) which, likeeIF5A1(K50A), cannot be hypusinated and, accordingly, is stronglyapoptogenic. The expression of eIF5AI(K50R) is preferably regulated by aB-cell-specific promoter.

The eIF5A1 siRNA is preferably specific to the 3′-end of endogenoushuman eIF5A1 and has no effect on expression of the trans eIF5A1(K50R).An exemplary preferred eIF5A1 siRNA comprises, consists essentially ofor consists of the siRNA shown in FIG. 25. The rationale for includingthe eIF5A1 siRNA is: (1) to deplete endogenous eIF5A1, which is almostall hypusinated and hence in the pro-survival form; (2) to inhibitactivation of NF-κB, and thereby reduce the production of IL-6 and theformation of intracellular adhesion molecules; and (3) to inhibit theformation of IL-1. That eIF5A1 siRNA acts synergistically witheIF5A1(K50R) to induce apoptosis in myeloma cells. Inasmuch as (2) and(3) above are pro-survival events, they are likely mediated byhypusinated eIF5A1, and hence not affected by eIF5A(K50R) which cannotby hypusinated. This approach results in a larger pool of unhypusinatedeIF5A leading to apoptosis of cancer cells, including multiple myelomacells, with little effect on healthy cells.

A preferred composition is referred to herein as SNS01. SNS01 is acomplex containing both, an RNAi-resistant plasmid DNA encodingeIF5A^(K50R) driven by a promoter that restricts expression to cells ofB-cell origin (including myeloma cells) for enhanced safety, and ansiRNA targeting human eIF5A with dTdT 3′ overhangs for enhanced nucleaseresistance and which the siRNA and the plasmid are complexed to in vivoJetPEI™.

EXAMPLES Example 1 Transfection of HeLaS3 Cells with Wild Type andVariants of eIF-5A1

HeLa S3 cells were transfected using Lipofectamine 2000 with plasmidsexpressing HA-tagged eIF5A1 variants including wild-type eIF5A1 (WT),eIF5A1K50R (K50R), eIF5A1K67R (K67R), eIF5A1K67A (K67A), eIF5A1K47R/K50R(K4750R), eIF5A1K50R/K67R (K5067R), or eIF5A1K50A/K67A (K5067A). Aplasmid expressing LacZ was used as a control. At 24 and 48 hours (A) or28 and 52 hours (B) after transfection, the cell lysate was harvestedand fractionated by SDS-PAGE. Expression levels of transfected eIF5A1was detected using an antibody against HA. Result: Mutation of eIF5A1 ata lysine in the putative ubiquination site (K67R) increased the 5accumulation of the eIF5A1 transgene above wild-type (A). Mutation ofeIF5A1 at the lysine required for hypusination (K50R) also increasedaccumulation of eIF5A1 transgene above wild-type eIF5A1 (B). A doublemutant form of eIF5A1 (K50A/K67A) was expressed particularly well whencompared to the unmutated wild-type eIF5A1 transgene (A+B). See FIGS. 2Aand 2B.

Example 2 Transfection of KAS Cells with Wild Type and Variants ofeIF-5A1

KAS cells were transfected using PAMAM dendrimer (FMD44) with plasmidsexpressing HA-tagged eIF5A1 variants including wild-type eIF5A1 (5A1),eIF5A1K67A (K67A), eIF5A1K50A/K67A (K50A K67A), eIF5A1K50R (K50R),eIF5A1K47R (K47R), eIF5A1K67R (K67R), eIF5A1K47R/K5OR (K47R K5OR), oreIF5A1K50R/K67R (K50R K67R). A plasmid expressing LacZ was used as acontrol. 48 hours (after transfection, the cell lysate was harvested andfractionated by SDS-PAGE. Expression levels of transfected eIF5A1 wasdetected using an antibody against HA. Equal loading was verified usingan antibody against actin. Result: Mutation of eIF5A1 at a lysine in theputative ubiquination site (K67A or K67R) increased the accumulation ofthe eIF5A1 transgene above wild-type. Mutation of eIF5A1 at the lysinerequired for hypusination (K5OR) or at an acetylation site (K47R) alsoincreased accumulation of eIF5A1 transgene above wild-type eIF5A. Adouble mutant form of eIF5A1 (K50A/K67A) was also expressed at higherlevels when compared to the unmutated wild-type eIF5A1 transgene. SeeFIG. 3.

Example 3 Transfection of KAS Cells Using PAMAM Dendrimer

KAS cells were transfected using PAMAM dendrimer (FMD45-2) with plasmidsexpressing HA-tagged eIF5A1 variants including eIF5A1K5OR (K5OR),eIF5A1K50A/K67A (K50A/K67A), or eIF5A1K50R/K67R (K50R K67R). A plasmidexpressing LacZ was used as a control. Seventy-two hours aftertransfection, the cells were stained with Annexin/PI and analyzed byFACS. Cells that stained positively for Annexin V and negatively for PI(propidium iodide) were considered to be in the early stages ofapoptosis (Ann+/PI−) and cells that stained positively for both AnnexinV and PI were considered to be in the late stages of apoptosis(Ann+/PI+). Result: Mutation of eIF5A1 at a lysine in the hypusinationsite (K50R) or in the putative ubiquination site (K67R), as well as thedouble mutant (K50A/K67A) resulted in apoptosis of KAS cellssignificantly above the levels of the LacZ control. See FIG. 4.

Example 4 Transfection of KAS Cells with Plasmids Expressing eIF-5A1 andeIF-5A1 Variants

KAS cells were transfected using Lipofectamine 2000 with plasmidsexpressing HA-tagged eIF5A1 variants including eIF5A1K50A (K50A),eIF5A1K50R (K50R), eIF5A1K67R (K67R), eIF5AI K50A/K67A (K50A/K67A), oreIF5A1K50R/K67R (K50R K67R). A plasmid expressing LacZ was used as acontrol. Seventy-two hours after transfection, the cells were stainedwith Annexin/PI and analyzed by FACS. Cells that stained positively forAnnexin V and negatively for PI (propidium iodide) were considered to bein the early stages of apoptosis (Ann+/PI−) and cells that stainedpositively for both Annexin V and PI were considered to be in the latestages of apoptosis (Ann+/PI+). Result: Mutation of eIF5A1 at a lysinein the hypusination site (K50R) or In the putative ubiquination site(K67R), as well as the double mutant (K50A/K67A) resulted in apoptosisof KAS cells significantly above the levels of the LacZ control. SeeFIG. 5.

Example 5 The Use of Mutated eIF-5A1 to Treat KAS Cells Results inApoptosis

KAS cells were transfected using Lipofectamine 2000 with plasmidsexpressing HA-tagged eIF5A1 variants eIF5A1K50R (K50R) oreIF5A1K50A/K67A (K50A/K67A. A plasmid expressing LacZ was used as acontrol. Seventy-two hours after transfection, the cells were stainedwith Annexin/PI and analyzed by FACS. Cells that stained positively forAnnexin V and negatively for PI (propidium iodide) were considered to bein the early stages of apoptosis (Ann+/PI−) and cells that stainedpositively for both Annexin V and PI were considered to be in the latestages of apoptosis (Ann+/PI+). Result: Mutation of eIF5A1 at a lysinein the hypusination site (K50R) or mutation of eIF5A1 at both thehypusination site and in the putative ubiquination site (K50A/K67A)resulted in apoptosis of KAS cells significantly above the levels of theLacZ control. See FIG. 6.

Example 6A siRNA/Adenovirus-Mediated Killing of Multiple Myeloma Cells

KAS (human multiple myeloma) cells were maintained in S10 media [RPMI1640 with 4 ng/ml IL-6, 10% fetal bovine serum (FBS), andpenicillin/streptomycin (P/S)]. KAS cells were transfected with 58.7pmoles of siRNA using Lipofectamine 2000 (Invitrogen). Mock transfectedcells were treated with Lipofectamine 2000 in the absence of siRNA.Transfection was conducted in the antibiotic-free S10 media.

a) siRNAs Targeting Human eIF5A1:

eIF5A1 siRNA target #1 (the siRNA targets this region of human eIF5A1:(SEQ ID NO: __) 5′-AAGCTGGACTCCTCCTACACA-3′.The siRNA sequence is shown in FIG. 25 and isoften referred to herein as h5A1.eIF5A1 siRNA target #2 eIF5A1 (this siRNA targetsthis region of human eIF5A1: (SEQ ID NO: __)5′-AAAGGAATGACTTCCAGCTGA-3′.(The siRNA sequence is often referred to herein ash5A1-ALT)b) control siRNA: The control siRNA had the following sequence:sense strand, 5′-ACACAUCCUCCUCAGGUCGdTdT-3′; and antisense strand,3′-dTdTUGUGUAGGAGGAGUCCAGC-5′″.

Other controls that have been used include non-targeting validatedsiRNAs from Dharmacon since they have been micro-array tested to limitunwanted off-targeting effects. For example, for in vitro work studyignNFkB, the control used was Dharmacon's non-targeting siRNA's (sequenceD-001700-01) and for in vivo work, the control used was Dharmacon's(sequence D-001810-01).

Four hours after transfection, the cells were pelleted and resuspendedin 1 ml of S10 media. Seventy-two hours after the initial siRNAtransfection, the transfected KAS cells were counted and seeded at300,000 cells/well in a 24-well plate and transfected with the samesiRNA a second time.

Four hours after transfection, the cells were pelleted and resuspendedin 1 ml of S10 media (without IL-6) containing 3000 ifu of eitherAd-LacZ (Adenovirus expressing B-galactosidase) or Ad-5A1M (Adenovirusexpressing human eIF5A1^(K50A)).

Seventy-two hours later the cells were harvested and analyzed forapoptosis by staining with Annexin V-FITC and PI (BD Bioscience)followed by FACS analysis.

-   -   a) early apoptosis was defined as cells that were positively        stained with Annexin-FITC and negative for PI-staining        (Ann+/PI−)    -   b) total apoptosis was defined as the total of cells either in        early apoptosis (Ann+/PI−) or late apoptosis/necrosis (Ann+/PI+)

The 5A1 siRNA targeting #1 targets the 3′UTR of human eIF5A1 andtherefore will not affect expression of eIF5A1 from adenovirus. 5A1siRNA targeting #2 targets within the open reading frame of human eIF5A1and so it could potentially interfere with expression of eIF5A1 from theadenovirus.

Results: Cells treated with siRNA and infected with adenovirusexpressing the eIF-5A1 K50A variant undergo apoptosis in greater numbersthan non-treated cells and cells treated only with siRNA. See FIG. 7.

Example 6B Pre-Treatment with eIF5A1 siRNA Against eIF5A1 Target #1(Shown in FIG. 25), Reduced Expression of Endogenous eIF5A1 but AllowsAccumulation of RNAi-Resistant eIF5A1^(k50A) Expressed by Adenovirus

KAS cells were transfected using Lipofectamine 2000 with either acontrol siRNA (C) or one of two siRNAs targeting eIF5A1 (#1 and #2). TheeIF5A1 siRNA #1 targets the 3′UTR of eIF5A1 and therefore does notinterfere with expression of eIF5A1 from adenovirus since it containsonly the open reading frame of eIF5A1. The sequence of the siRNA isshown in FIG. 25. The eIF5A1 #2 siRNA targets the open reading frame ofeIF5A1 and will therefore affect expression of both endogenous andexogenously-expressed eIF5A1. Seventy-two hours after the initialtransfection hours the cells were transfected with the same siRNA asecond time. Four hours later the transfection complexes were removedfrom the cells and replaced with growth media (−) IL6 containing eitherAd-LacZ (L) or Ad-eIF5A^(K50A) (5M). Seventy-two hours later the celllysate was harvested and analyzed by Western blot using antibodiesagainst eIF5A and actin. See FIG. 7B. Accumulation of virally expressedeIF5A1 can be observed (lane 1 vs lane 2) and reduction of eIF5Aexpression by eIF5A1 siRNAs targeting #1 and #2 can be clearly seen(lanes 5 and 7 vs lane 3). As expected, the eIF5A1 siRNA #1 does notaffect accumulation of the virally expressed eIF5A1^(K50A) (lane 6 vslane 4) while the eIF5A1 siRNA #2 only moderately affects expression ofthe virally-expressed transgene (lane 8 vs lane 4).

Example 6C Pre-Treatment with eIF5A1 siRNA Against Target #1 Prior toAdenovirus Infection Reduces Expression of Phosphorylated NF-κB in HumanMultiple Myeloma Cells

KAS cells were transfected using Lipofectamine 2000 with either acontrol siRNA (hC) or an siRNA targeting eIF5A1 (#1). The eIF5A1 siRNA#1 targets the 3′UTR of eIF5A1 and will therefore not interfere withexpression of eIF5A1 from adenovirus since it contains only the openreading frame of eIF5A1. Seventy-two hours after the initialtransfection hours the cells were transfected with the same siRNA asecond time. Four hours later the transfection complexes were removedfrom the cells and replaced with growth media (+) IL6 containing eitherAd-LacZ (L) or Ad-eIF5A1^(K50A) (5M). Twenty-four hours later the celllysate was harvested and analyzed by Western blot using antibodiesagainst phospho-NF-kB p65 (Ser 536) and eIF5A. As expected, the eIF5A1siRNA #1 does not affect accumulation of the virally expressedeIF5A1^(K50A). Phosphorylation of NF-kB p65 at serine 536 regulatesactivation, nuclear localization, protein-protein interactions, andtranscriptional activity. See FIG. 7C.

Example 6D Pre-Treatment with eIF5A1 siRNA #1 Prior to AdenovirusInfection Reduces Expression of Phosphorylated NF-kB and ICAM-1 in HumanMultiple Myeloma Cells

KAS cells were transfected using Lipofectamine 2000 with either acontrol siRNA (C) or one of two siRNAs targeting eIF5A1 (#1 and #2).Seventy-two hours after the initial transfection hours the cells weretransfected with the same siRNA a second time. Four hours later thetransfection complexes were removed from the cells and replaced withgrowth media (+) IL6. Twenty-four hours after the second transfection,the cells were stimulated with 40 ng/ml TNF-α and cell lysate washarvested at 0, 4, or 24 hours and analyzed by Western blot usingantibodies against phospho-NF-kB p65 (Ser 536), ICAM-1 and actin. Areduction in TNF-α induced NF-kB p65 phosphorylation (ser 536) andICAM-1 expression was observed following transfection with botheIF5A1-specific siRNAs. Phosphorylation of NF-kB p65 at serine 536regulates activation, nuclear localization, protein-proteininteractions, and transcriptional activity. ICAM-1 is an inter-cellularadhesion surface glycoprotein that is believed to be involved in thepathogenesis of multiple myeloma. See FIG. 7D.

Example 6E Pretreatment of KAS Cells with siRNA Increases Apoptosis byeIF5A1^(k50R) Gene Delivery in the Presence of IL-6

KAS cells were transfected with either control siRNA (hcon) or humaneIF5A1 siRNA (h5A1) using Lipofectamine 2000. Seventy-two hours laterthe cells were re-transfected with siRNA. PEI complexes of empty vector(mcs) or eIF5A1^(k50R) (K50R) plasmids were added to the cells fourhours later following removal of siRNA transfection medium. The growthmedium used throughout the study contained IL-6. Apoptosis was measuredseventy-two hours later by staining the cells with Annexin/PI and FACSanalysis. See FIG. 7F.

Example 7 Co-Administration of eIF-5A1 Plasmid and eIF-5A1 siRNA DelaysGrowth of Multiple Myeloma Subcutaneous Tumors (FIGS. 8-10)

SCID mice were injected subcutaneously with KAS cells. Treatment wasinitiated when palpable tumours were observed. Six 3-5 week oldSCID/CB17 mice were injected with 10 million KAS-6/1 myeloma cells in200 μL PBS in their right flank and treatment was initiated when thetumours reached a minimum size of 4 mm³.

Control mice were injected intra-tumourally 2 times per week with PEIcomplexes containing pCpG-mcs (empty vector) and control siRNA (controlgroup was made up of 3 mice: C-1, C-2, and C-3). Treated mice wereinjected intra-tumourally 2 times per week with PEI complexes containingthe RNAi-resistant plasmid pCpG-eIF5A1k50R and eIF5A1 siRNA (treatedgroup was made up of 3 mice: 5A-1, 5A-2, and 5A-3). Injections weregiven at multiple sites within the tumor to prevent reflux and a slowrate of injection was used to increase uptake. The data in FIG. 8 showsthe tumor volume for all the mice in the group. The data shown in FIG. 9is the average tumor volume per group +/−standard error. Asterix denotestatistical significance (*=p<0.025; n=3).

FIG. 10 shows that co-administration of eIF-5A1 plasmid and eIF-5A1siRNA reduces the weight of multiple myeloma subcutaneous tumors. Thedata shown is the average tumor weight per group +/−standard error.Asterix denote statistical significance (*=p<0.05; n=3).

JET-PEI (PolyPlus) at 2×0.1 ml was used for the in vivo tests. The N/Pratio was 8. The PEI/DNA/siRNA complexes were formed in a total volumeof 0.1 ml in 5% glucose. The protocol for forming complexes was asfollows.

-   -   1. Bring components to room temperature. Keep sterile.    -   2. Dilute 20 μg of plasmid DNA (˜10 μl at 2 mg/ml) and 10 μg        siRNA (10 μl at 1 mg/ml) into a total volume of 25 μl. Use        sterile water to make up difference.    -   3. Adjust the volume of DNA solution to 50 μl 5% glucose by        adding 25 μl of 10% glucose. Vortex gently and centrifuge        briefly.    -   4. Dilute 4.8 μl of in vivo JETPEI into a total volume of 25 μl        of 10% glucose. Adjust volume to 50 μl with sterile water to end        up with a final concentration of 5% glucose. Vortex gently and        centrifuge briefly.    -   5. Immediately add 50 μl of diluted PEI to the 50 μl of diluted        DNA (do not reverse the order). Vortex briefly and immediately        spin down.    -   6. Incubate for 15 minutes prior to injection. Complexes are        stable for 6 hours.

CpG-free Cloning Vectors and pCpG Plasmids were obtained from InvivoGen.These plasmids are completely devoid of CpG dinucleotides, named pCpG.These plasmids yield high levels of transgene expression both in vitroand in vivo, and in contrast to CMV-based plasmids allow sustainedexpression in vivo. pCpG plasmids contain elements that either naturallylack CpG dinucleotides, were modified to remove all CpGs, or entirelysynthesized such as genes encoding selectable markers or reporters.Synthesis of these new alleles was made possible by the fact that amongthe sixteen dinucleotides that form the genetic code, CG is the onlydinucleotide that is non-essential and can be replaced. Eight codonscontain a CG encoding for five different amino acids. All eight codonscan be substituted by at least a choice of two codons that code for thesame amino acid to create new alleles that code for proteins havingamino acid sequences that remain identical to the wild type and thus areas active as their wild-type counterparts. These new alleles areavailable individually in a plasmid named pMOD from which they can beeasily excised.

pCpG plasmids allow long lasting expression in vivo, and representvaluable tools to study the effects of CpGs on gene expression in viveand in vitro, using cell lines expressing TLR9, as well as their effectson the innate and acquired immune systems.

The empty vector, pCpG-mcs (Invivogen) is a vector with no expressedgene product, only a multiple cloning site, and was used as the controlvector. An HA-tagged eIF5A1^(k50R) cDNA was subcloned into the NcoI andNheI sites of a pCpG-LacZ vector (Invivogen), from which the LacZ genehad been removed, to give rise to the treatment vectorpCpG-eIF5A1(K50R). The DNA was prepared using Endo-Free Qiagen kit.Endotoxin levels measured and are <0.03 EU/ug; DNA should be at 2 mg/mlin water.

The control siRNA used in the experiments was a micro-array validatednon-targeting control siRNA from Dharmacon (D-001810-01). The siRNA wasobtained with a modification (siSTABLE) to prevent degradation in serum.

The eIF5A1 siRNA used in the experiments was designed against the 3′UTRof human eIF5A1. There is no similarity between the eIF5A1 siRNA andmouse eIF5A1 and the siRNA should therefore only suppress human (but notmouse) eIF5A1. The siRNA also has no similarity to eIF5A2 (either humanor mouse). The siRNA was obtained with a modification (siSTABLE) toprevent degradation in serum. The eIF5A1 siRNA has the following targetsequence:

5′ GCU GGA CUC CUC CUA CAC A (UU) 3

The siSTABLE siRNA was dissolved at 1 mg/ml in water (stored in aliquotsat −20 C).

Tumour dimensions of length (l) and width (w) were measured 2-3 timesper week using digital calipers. Tumour volume was calculated accordingto the following equation:

l=length; smallest dimension

w=width; largest dimension

tumour volume (mm³)=l ² *w*0.5

Statistical Analyses

Student's t-test was used for statistical analysis. Significance wasdeemed to be a confidence level above 95% (p<0.05).

Example 8 Co-Administration of eIF5A1 Plasmid and eIF5A1 siRNA DelaysGrowth of Multiple Myeloma Subcutaneous Tumors and Results in TumorShrinkage

In another study, again SCID mice were injected subcutaneously with KAScells. Treatment was initiated when palpable tumours were observed.Control mice were injected intra-tumourally 2 times per week with PEIcomplexes containing pCpG-mcs (empty vector) and control siRNA (controlgroup G-1, G-2 and G-3). Treated mice were injected intra-tumourally 2times per week with PEI complexes containing the RNAi-resistant plasmidpCpG-eIF5A1k50R (20.g of plasmid DNA) and eIF5A1 siRNA (10 g of siRNA)(treated group G-4, G-5 and G-6). The data shown in FIG. 11 is theaverage tumour volume per group +/−standard error. Asterix denotestatistical significance (*=p<0.025; n=3). Six injections over a periodof 21 days were given (red arrows).

Example 9 Administration of eIF5A1 siRNA Intra-Venously (i.v.) andPEI/eIF5A1K50R Plasmid Complexes Intra-Tumourally (i.t.) Results inTumour Shrinkage of Multiple Myeloma Subcutaneous Tumours (Group 2B)

SCID mice were injected subcutaneously with KAS cells. When palpabletumours were observed treatment was initiated with an initial tailinjection of 50 micrograms of either control siRNA (control group) orhuman eIF5A1 siRNA (treated group). Control Mice were subsequentlytreated by intra-tumoural injections 2 times per week with PEI complexescontaining pCpG-ms (empty vector, control group; G-1, G-2, and G-3).Treated mice were subsequently treated by intra-tumoural injections 2times per week with PEI complexes containing the RNAi-resistant plasmidpCpG-eIF5A1k50R (20 g plasmid DNA) (treated group; G-4, G-5, and G-6).Control mice continued to receive control siRNA (control group R-1, R-2,and R-3) by i.v. injection once per week. Treated mice continued toreceive human eIF5A1 siRNA (20 μg) (treated group R-4, R-5, and R-6) byi.v. injection once per week. The data shown in FIG. 12 is the tumorvolume for all the mice in each group. Six intramural injections ofPEI/DNA (red arrows) and four i.v. injections of siRNA (blue arrows)were given over a period of 21 days.

FIG. 13 provides an overlay of the results from Example 8 and 9. SCIDmice were injected subcutaneously with KAS cells. The data shown in FIG.13 is the average tumor volume for the mice in each group +/−standarderror. Asterix denote statistical significance between treated andcontrol groups (**=p<0.01; ***=p<0.001; n=3).

Protocol for Forming PEI Complexes:

-   -   1. Bring components to room temperature. Keep sterile.    -   2. Dilute plasmid DNA or plasmid DNA+siRNA into a total volume        of 25 μl. Use sterile water to adjust the volume.    -   a) For plasmid DNA only complexes:        -   Dilute 20 μg of plasmid DNA (10 μl at 2 mg/ml) into a total            volume of 25 μl. Use sterile water to make up difference.    -   b) For plasmid DNA+siRNA complexes:        -   Dilute 20 μg of plasmid DNA (−10 μl at 2 mg/ml) and 10 g of            siRNA (10 μl at 1 mg/ml) into a total volume of 25 μl. Use            sterile water to make up difference.    -   3. Adjust the volume of DNA solution to 50 μl 5% glucose by        adding 25 μl of 10% glucose (provided with PEI kit). Vortex        gently and centrifuge briefly.    -   4. Dilute in vivo JETPEI into a total volume of 25 μl of 10%        glucose.    -   a) For plasmid DNA only complexes:        -   Dilute 3.2 μl of in vivo JETPEI into a total volume of 25 μl            of 10% glucose. Adjust volume to 50 μl with sterile water to            end up with a final concentration of 5% glucose. Vortex            gently and centrifuge briefly.    -   b) For plasmid DNA+siRNA complexes:        -   Dilute 4.8 μl of in vivo JETPEI into a total volume of 25 μl            of 10% glucose. Adjust volume to 50 μl with sterile water to            end up with a final concentration of 5% glucose. Vortex            gently and centrifuge briefly.    -   5. Immediately add 50 μl of diluted PEI to the 50 μl of diluted        DNA (do not reverse the order!). Vortex briefly and immediately        spin down.    -   6. Incubate for 15 minutes prior to injection. Complexes are        stable for 6 hours.

Regarding the tail-vein injection of siRNA, the initial siRNA injectionwas 50 micrograms. siRNA was diluted to 0.4 mg/ml in PBS. 125 μl permouse (50 μg) was injected into the tail vein. Subsequent injections ofserum-stabilised siRNA were given two times per week at 20 μg per mouse.siRNA was diluted to 0.4 mg/ml in PBS. 50 μl per mouse (20 μg) wasinjected into the tail vein.

FIG. 13B shows that co-administration of eIF5A1 plasmid and eIF5A1 siRNAresults in tumour shrinkage. SCID mice were injected subcutaneously withKAS cells. Treatment was initiated when palpable tumours were observed.Mice were injected intra-tumourally 2 times per week with PEI complexescontaining the RNAi-resistant plasmid pCpG-eIF5A1k50R and eIF5A1 siRNA(treated group; 0-4, G-5, and G-6). Six injections over a period of 21days were given. Forty-two days after the initiation of treatment themice were sacrificed and the skin under the tumour site was opened andexamined for evidence of tumour growth. No tumour growth was observed inany of the group 2A treated mice.

FIG. 13C shows that administration of eIF5A 1 siRNA intra-venously(i.v.) and PEI/eIF5A1K50R plasmid complexes intra-tumourally (i.t.)results in tumour shrinkage of multiple myeloma subcutaneous tumours.SCID mice were injected subcutaneously with KAS cells. When palpabletumours were observed treatment was initiated with an initial injectionof 50 micrograms of human eIF5A1 siRNA (treated group). Mice weresubsequently treated by intra-tumoural injections 2 times per week withPEI complexes containing the RNAi-resistant plasmid pCpG-eIF5A1k50R(treated group; R-4, R-5, and R-6). Mice continued to receive humaneIF5A1 siRNA by i.v. injection once per week. Treatment ended 21 daysafter initiation of treatment. Forty-two days after the initiation oftreatment the mice were sacrificed and the skin under the tumour sitewas opened and examined for evidence of tumour growth. There was noevidence of tumour growth in one mouse of the treatment group (group2B).

Example 10 Intra-Venous Co-Administration of eIF5A1 Plasmid and eIF5A1siRNA Delays Growth of Multiple Myeloma Subcutaneous Tumours

SCID mice were injected subcutaneously with KAS cells. When palpabletumours were observed treatment was initiated with an initial injectionof 50 micrograms of either control siRNA (control group) or human eIF5A1siRNA (treated group). Mice were subsequently treated by intra-venous(red arrows) or intra-peritoneal injections (green arrow)˜twice per weekwith either PEI complexes containing pCpG-mcs (empty vector; controlgroup A1, A2, and A3) or PEI complexes containing the RNAi-resistantplasmid pCpG-eIF5A1k50R (treated group; A4, A5, and A6). Mice continuedto receive either control siRNA (control group A1, A2, and A3) or humaneIF5A1 siRNA (treated group A4, A5, and A6) by i.v. injection (bluearrows) once per week. The data shown is the tumour volume for all themice in each group. The data shown in FIG. 14 is the tumour volume forall the mice in each group.

Example 11 Administration of eIF5A1 siRNA Intra-Venously (i.v.) andPEI/eIF5A1K50R Plasmid Complexes Intra-Venously (i.v.) orIntra-Peritoneal (i.p.) Delays Growth of Multiple Myeloma SubcutaneousTumours

SCID mice were injected subcutaneously with KAS cells. When palpabletumours were observed treatment was initiated with an initial injectionof 50 micrograms of either control siRNA (control group) or human eIF5A1siRNA (treated group). Control mice were subsequently treated byintra-venous or intra-peritoneal injections once per week with PEIcomplexes containing pCpG-mcs (empty vector; control group was threemice: B1, B2, and B3). Treated mice were subsequently treated byintra-venous or intra-peritoneal injections˜once per week with PEIcomplexes containing the RNAi-resistant plasmid pCpG-eIF5A1^(K50R)(treated group; B4, B5, and B6). Mice continued to receive eithercontrol siRNA (control group B1, B2, and B3) or human eIF5A1 siRNA(treated group was three mice: B4, B5, and B6) by i.v. injection onceper week. The experiment began with initial siRNA injection of 50 μg(day-2 on graph in FIG. 15). Subsequent injections used 20 micrograms ofsiRNA once weekly. The siRNA was given naked, i.e. no delivery vehicle.PEI complexes contained 20 μg of plasmid DNA. The initial PEI injectionwas given i.p. and subsequent injections were given i.v. The data shownin FIG. 15 is the tumor volume for all the mice in each group.

FIG. 16 provides an overlay of Example 10 and 1. SCID mice were injectedsubcutaneously with KAS cells. Treatment was initiated when palpabletumours were observed. One set of mice received i.v. injections ofeither control siRNA (control; Group A) or eIF5A1 siRNA (treated; GroupA) once per week and either i.v. or i.p. of either PEI complexescontaining pCpG-mcs (control; Group A) or PEI complexes containing theRNAi-resistant plasmid pCpG-eIF5A1^(K50R) (treated; Group A). A secondset of mice were given i.v. or i.p. injections˜2 times per week witheither PEI complexes containing pCpG-mcs (empty vector) and controlsiRNA (control; Group B.) or PEI complexes containing the RNAi-resistantplasmid pCpG-eIF5A1k50R and eIF5A1 siRNA (treated; Group B). The datashown is the average tumour volume for the mice in each group +/−5standard error. Asterix denote statistical significance between treatedand control groups (*=p<0.05; ***=p<0.001; n=3).

The protocols for preparing the PEI complexes and the siRNA are asdescribed in previous examples.

Example 12 Co-Administration of eIF5A Plasmid and eIF5A1 siRNA DelaysGrowth of Multiple Myeloma Subcutaneous Tumours and Results in TumourShrinkage

SCID mice were injected subcutaneously with KAS cells. Treatment wasinitiated when palpable tumours were observed. Control mice wereinjected intra-tumourally 2 times per week with PEI complexes containingpCpG-mcs (empty vector) and control siRNA (control group had 3 mice:control 1, control 2, and control 3). Treated mice were injectedintra-tumourally 2 times per week with PEI complexes containing theRNAi-resistant plasmid pCpG-eIF5A1^(K50R) and eIF5A1 siRNA (treatedgroup contained 4 mice: 5A-1, 5A-2, 5A-3, and 5A-4). The intra-tumoralrejections of PEI complexes contained both 20 ug of plasmid DNA and 10μg of siRNA. The data shown in FIGS. 17A and 17B is the tumour volumefor all the mice in each group.

Example 13 Administration of eIF5A1 siRNA Intra-Venously (i.v.) andPEI/eIF5A1^(K50R) Plasmid Complexes Intra-Tumourally (i.t.) Results inTumour Shrinkage of Multiple Myeloma Subcutaneous Tumours

SCID mice were injected subcutaneously with KAS cells. When palpabletumours were observed treatment was initiated with an initial injectionof 50 micrograms of either control siRNA (control group had three mice:control 1, control 2 and control 3) or human eIF5A1 siRNA (treated grouphad 3 mice: 5A-1, 5A-2, 5A-3). Control mice were subsequently treated byintra-tumoural injections 2 times per week with PEI complexes containingpCpG-mcs (20 μg) (control group 1-3). Treated mice were subsequentlytreated by intra-tumoural injections 2 times per week with PEI complexescontaining the RNAi-resistant plasmid pCpG-eIF5A^(K50R) (20 μg) (5A-1,5A-2, 5A-3). Control mice continued to receive either control siRNA (20μg) by tail vein i.v. injection twice per week. Treated mice continuedto receive human eIF5A1 siRNA (20 μg) by tail vein i.v. injection twiceper week. The injections were give 48 hours prior to the intra-tumouralinjections. The siRNA was given as naked siRNA, i.e. no deliveryvehicle. The data shown in FIGS. 18A and 18B is the tumour volume forall the mice in each group.

Example 14 Co-Administration of eIF5A1^(K50R) Plasmid, Driven by Eitherthe EF1 or B29 Promoter, and eIF5A1 siRNA Delays Growth of MultipleMyeloma Subcutaneous Tumours and Results in Tumour Shrinkage

SCID mice were injected subcutaneously with KAS cells. Treatment wasinitiated when palpable tumours were observed. Mice were injectedintra-tumourally 2 times per week with PEI complexes containing eithercontrol vector (G1 and G2) or an eIF5A1 plasmid driven by either the B29promoter (G3 and G4) or the EF1 promoter (G5 and G6) and either controlsiRNA (G1, G3, G5) or h5A1 siRNA (G2, G4, G6). The data shown is theaverage tumour volume +/−standard error for each group. Note: the B29promoter was intended as a B-cell-specific promoter. However, althoughthe B29 promoter/mCMV enhancer used in this study was found to drivehigh expression of HA-eIF5A1^(K)50R in KAS cells in vitro, it does notappear to be B-cell-specific (likely due to CMV enhancer). See FIG. 19.

Example 15 Co-Administration of eIF5A1 siRNA Increases Anti-Tumor Effectof eIF5A1^(K50R) Plasmid, Driven by Either the EF1 or B29 Promoter, onMultiple Myeloma Subcutaneous Tumours and Results in Reduced TumorBurden

SCID mice were injected subcutaneously with KAS cells. Treatment wasinitiated when palpable tumours were observed. Mice were injectedintra-tumourally 2 times per week with PEI complexes containing eithercontrol vector (G1 and G2) or an eIF5A1 plasmid driven by either the B29promoter (G3 and G4) or the EF1 promoter (G5 and G6) and either controlsiRNA (G1, G3, G5) or h5A1 siRNA (G2, G4, G6). The mice were sacrificed24 days after the initiation of treatment and the subcutaneous tumor wasremoved and weighed. The data shown is the average tumor weight+/−standard error for all groups. See FIG. 20.

Example 16 eIF5A1 siRNA Synergistically Increases Apoptosis InductionResulting from Infection with Ad-eIF5A in Lung Adenocarcinoma Cells

A549 cells were infected with either Ad-LacZ or Ad-eIF5A. Cells weretransfected with either a control siRNA or an siRNA targeting humaneIF5A1 (h5A1) by adding the transfection media to cells immediatelyfollowing addition of the virus. Four hours after transfection with thesiRNA and infection with virus, the media was replaced with fresh mediaand the cells were incubated for 72 hours prior to labelling withAnnexin/PI to detect apoptotic cells. Note: over-expression of eIF5A inthis cell line results in the accumulation of unhypusinated eIF5A due tolimiting amounts of DHS and DOHH and therefore results in samepro-apoptotic effect as over-expression of eIF5A^(K50R). These dataindicate that the synergistic effect in apoptosis caused by simultaneoussuppression of hypusinated eIF5A and over-expression of unhypusinatedeIF5A is observed in non-myeloma tumour-types as well. See FIG. 21.

Example 17 Construction of Plamsid pExp5A

pExp5A is an expression plasmid with reduced CpG dinucleotides designedto drive expression of human eIF5A1^(K50R) predominantly in cells of Bcell lineage. The vector is derived from pCpG-LacZ, a plasmid completelydevoid of CpG dinucleotides (Invivogen). All the elements required forreplication and selection in E. coli are free of CpG dinucleotides. Theoriginal CMV enhancer/promoter and LacZ gene from the CpG-LacZ vectorhave been replaced with a human minimal B cell specific promoter(B29/CD79b; Invivogen) and human eIF5A1^(K50R), respectively, in orderto drive B-cell specific expression of eIF5A1^(K50R). The B29 DHS4.4 3′enhancer has been introduced into the plasmid downstream of the eIF5A1expression cassette in order to enhance activity of the B29 promoter andreduce expression in non-B cells (Malone et al. 2006. B29 gene silencingin pituitary cells is regulated by its 3′ enhancer. J. Mol. Biol. 362:173-183). Incorporation of the B29 minimal promoter, eIF5A1^(K50R), andthe B29 DHS4.4 3′ enhancer has introduced 32 CpG dinucleotides into thevector.

Elements for expression in E. coliOrigin of replication: E. coli R6K gamma ori.*Due to the presence of the R6K gamma origin of replication, pCpGplasmids can only be amplified in E. coli mutant strain expressing a pirmutant gene. They will not replicate in standard E. coli strains.Therefore, pCpG plasmids are provided with the E. coli GT115 strain, apir mutant also deficient in Dcm methylation (Invivogen).Bacterial promoter: EM2K, a CpG-free version of the bacterial EM7promoter.Selectable marker: Zeocin™ resistance gene; a synthetic allele with noCpGs.

Elements for Expression in Mammalian Cells

Mammalian promoter: the human −167 bp minimal B29 (CD79b) promoter fortissue-specific expression in B cells. A synthetic intron (I 140) isalso present in the 5′UTR. Polyadenylation signal: a CpGdinucleotide-free version of the late SV40 polyadenylation signal.3′ Enhancer: the human B29 DHS4.4 3′ enhancer.MAR: Two CpG-free Matrix attached regions (MAR) are present between thebacterial and mammalian transcription units. One MAR is derived from the5′ region of the human IFN-f3 gene and one from the 5′ region of the3-globin gene.

The predicted Sequence of pExp5A (3371 bp is provided at FIGS. 23A and23B.

Amino Acid Sequence of eIF5A1^(K50R)MADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCKIVEMSTSKTGRFIGHAKVHLVGIDIFTGKKYEDICPSTHNMDVPNIKRNDFQLIGIQDGYLSLLQDSGEVREDLRLPEGDLGKEIEQKYDCGEEILITVLSAMTE EAAVAIKAMAK* K50R mutation is underlinedConstruction of pExp5A—Outline of Construction:Step 1: Cloning of B29 DHS4.4 3′ enhancer and subcloning into pGEM Teasy (Promega)—creates pGEM-4.4enh #8.Step 2: Subcloning of minimal B29 promoter into pCpG-LacZ(Invivogen)—creates B29-5 #3.Step 3: Subcloning of HA-eIF5A10R into B29-5#3 vector—createsB29-5#3-eIF5A1^(K50R).Step 4: Creation of new multiple cloning site in pCpG-mcs(Invivogen)—creates pCpG-Linker4.Step 5: Subcloning of B29 DS4.4 3′ enhancer into pCpG-Linker-4—createspCpG-DHS4.4.Step 6: Subcloning of B29 promoter+HA-eIF5A1^(K50R)+SV40 pA expressioncassette into pCpG-DHS4.4 creates pExp-5.Step 7: Replacement of HA-eIF5A1^(K50R) in pExp-5 with non-HA eIF5A1 KORcreates final vector, pExp5A.

Construction in Detail:

Step 1: Cloning of B29 DHS4.4 3′ Enhancer and Subcloning into pGEM TEasy (Promega)—Creates pGEM-4.4Enh #8.

The B29 DHS4.4 3′ enhancer was cloned by PCR from genomic DNA isolatedfrom KAS cells (human multiple myeloma cell line) using the followingprimers: forward 5′-CAGCAAGGGAGCACCTATG-3′ and reverse5′-GTTGCAGTGAGCGGAGATG-3′. The primers were designed using the sequenceof the human CD79B/GH-1 Intergenic region (Accession AB062674). Theresulting 608 bp PCR fragment was subcloned into the pGEM® T easycloning vector (Promega) and sequenced. Komatsu et al. 2002. Novelregulatory regions found downstream of the rat B29/Ig-b gene. Eur. J.Biochem. 269: 1227-1236.

Sequence of B29 DHS4.4 3′ enhancer PCR fragment(297 bp) in pGEM-4.4enh #8

+4.4 regions contains several transcription factor binding sites

Alignment of B29 DHS4.4 3′enhancer PCR fragment (297 bp) in pGEM-4.4enh #8with sequence of the human CD79B/GH-1 Intergenic region (Accession AB062674).1       10        20        30        40        50        60        70|--------+---------+---------+---------+---------+---------+---------+

GGGA

CAGCTGCCAGCTGGGAGACCAAGTGC

TC

CCT

CGTGCA

C

TCCCT

CC

CCAGC PCR Consensus**********************************************************************        80        90       100       110       120       130---------+---------+---------+---------+---------+---------|

CTGTGCTCCACTTCCTGTTGACCCTGG

GGG

TCCTTCGAGGCCCCTCTGCTATTCCT

PCR Consensus************************************************************131    140       150       160       170       180       190       200|--------+---------+---------+---------+---------+---------+---------+

CTCTGAATTCCAGCAGGGGAGCACCTATGCTGTGGGAGCTGCCAGTTT

TGGGGA

TCA

GA

CAGCA PCR Consensus**********************************************************************       210       220       230       240       250       260---------+---------+---------+---------+---------+---------|

CAGGGGAACTAGTG

G

AC

GTGCCAATTTTC

CC

TTCCCTCTG

TTCC

GGTGG PCR Consensus************************************************************261    270       280       290       300       310       320       330|--------+---------+---------+---------+---------+---------+---------+

GGCAGGTGGGTA

GGCCCCC

CGCCTGCAGTTTCAGGT

TCTCTCCACC

PCR                                                 ACC

Consensus ************************************************ACC

       340       350       360       370       380       390---------+---------+---------+---------+---------+---------|

PCR

Consensus

391    400       410       420       430       440       450       460|--------+---------+---------+---------+---------+---------+---------+

PCR

Consensus

       470       480       490       500       510       520---------+---------+---------+---------+---------+---------|

PCR

Consensus

521    530       540       550       560       570       580       590|--------+---------+---------+---------+---------+---------+---------+

PCR

Consensus

       600       610       620       630       640       650---------+---------+---------+---------+---------+---------|

       ACCTCTG

ACACAGTTTCCCTGAGACTTTGA

GCTCTTGTTTTATTTA PCR

       ACCTCT Consensus

       ACCTCT*********************************************651    660       670       680       690       700       710       720|--------+---------+---------+---------+---------+---------+---------+

TTTATTTATTTATTTATTTACTTATTTATTTATTTGCAGACAGAGTCTCACTCTGTTGCCCAGACTG

PCR Consensus**********************************************************************       730       740       750       760       770       780---------+---------+---------+---------+---------+---------|

TGCAGTGGCACCATCTCCGCTCACTGCAACCTCCGTCTCCTGAGTTCAAGCAATTCTCCT PCRConsensus ************************************************************781    790       800 |--------+---------|

GCCTCAGCCTCCAAAGTACC PCR Consensus ********************

indicates data missing or illegible when filedStep 2: Subcloning of Minimal B29 Promoter into pCpG-LacZ(Invivogen—Creates B29-5 #3

The minimal −167 human B29 promotor was amplified from a commercialplasmid bearing the full length human B29 promotor (pDrive-hB29;Invivogen) using the following primers: forward5′-CCAACTAGTGCGACCGCCAAACCTTAGC-3′; reverse:5′-CAAAAGCTTGACAACGTCCGAGGCTCCTTGG-3′. The resulting PCR fragment wasdigested with SpeI and HindIII and ligated into the SpeI and HindIIIsites of the pCpG-LacZ vector (Invivogen) to create B29-5 #3.

Sequence of B29 minimal promoter PCR fragment (188 bp) in B29-5 #3GCGACCGCCAAACCTTAGCGGCCCAGCTGACAAAAGCCTGCCCTCCCCCAGGGTCCCCGGAGAGCTGGTGCCTCCCCTGGGTCCCAATTTGCATGGCAGGAAGGGGCCTGGTGAGGAAGAGGCGGGGAGGGGACAGGCTGCAGCCGGTGCAGTTACACGTTTTCCTCCAAGGAGCCTCGGACGTTGTCAlignment of B29 minimal promoter PCR fragment (B29_min) in B29-5 #3 with fulllength human B29 promoter from pDrive-hB291       10        20        30        40        50        60        70|--------+---------+---------+---------+---------+---------+---------+B29_promCCTGCAGGGCCCACTAGTAAACGGAGGGTTGTAAGGAGAGTGAGAGGTGGACAGAGGGCACCGACGATTTB29_min Consensus**********************************************************************        80        90       100       110       120       130---------+---------+---------+---------+---------+---------| B29_promAGCATCTCTTCCTCTCCTGGGGGTCA

GGATGAGAGACAAAAAGAAGCTGCCAGG

AAAC B29_min Consensus************************************************************131    140       150       160       170       180       190       200|--------+---------+---------+---------+---------+---------+---------+B29_prom ATA

ATTCAGAGGGCTC

CTGC

CTGAGGTCTGCAAGCATGCTGTGTACACTTGT

CATGTTGTT B29_min Consensus**********************************************************************       210       220       230       240       250       260---------+---------+---------+---------+---------+---------| B29_promCCCTGCACAAGGGCATCTCTGAAGGGGCTGCACTGGACCCA

AGGGGCGCAAAGGT B29_min Consensus************************************************************261    270       280       290       300       310       320       330|--------+---------+---------+---------+---------+---------+---------+B29_prom GAGTTTATATCAGTTCCTGAGCACTGTGGCTCCATCCAGCACTCTGAGGAC

A

ATACAGCTGGAG B29_min Consensus**********************************************************************       340       350       360       370       380       390---------+---------+---------+---------+---------+---------| B29_promGACCTGAGGGCT

A

A

AGCTCCTGTTCCCTGCCC

AGACCCCCTGGACCTGCAG B29_min Consensus************************************************************391    400       410       420       430       440       450       460|--------+---------+---------+---------+---------+---------+---------+B29_prom ACAACAATTCAACGCACTCAGAGTCCCACAGTTAGGAACTCCCTG

GCCCCCAGTGGCTGCGT

T B29_min Consensus**********************************************************************       470       480       490       500       510       520---------+---------+---------+---------+---------+---------| B29_promGGATTTTCGCA

GCTGTCTCCACCTACATCCACCCTGTTTGGCAGCCCCTACATACTCT B29_min Consensus************************************************************521    530       540       550       560       570       580       590|--------+---------+---------+---------+---------+---------+---------+B29_prom TTCA

CATGAGGAAGGGAGGCCTCTC

CC

AGACCTGG

CT

TCTTCTCCCAGTGGCTGCCAC

CC B29_min Consensus**********************************************************************       600       610       620       630       640       650---------+---------+---------+---------+---------+---------| B29_promTGACCTGCTCTTGCTCC

CCTCTGTGGCTCCC

CCACAGGGTCAACTTCCAAC B29_min Consensus************************************************************651    660       670       680       690       700       710       720|--------+---------+---------+---------+---------+---------+---------+B29_prom ATGGCTGCCTGCACTCCAGCCAAGAGGCTCTGCTCTGGGCCCCTCCAGATGCCT

CCTGGGTCTGT

C B29_min Consensus**********************************************************************       730       740       750       760       770       780---------+---------+---------+---------+---------+---------| B29_promTGCCCTGTCCTTCTTCAGTGCTCCTCTTCCCGCTGGGTG

GGAATAGTTCAGGAC

B29_min Consensus************************************************************781    790       800       810       820       830       840       850|--------+---------+---------+---------+---------+---------+---------+B29_prom

GCTAAGTTCAGGTTCATTC

TAGGACAGGTGCCTATTTCGCTCACGGCCC

T

GACTTGCCGG B29_min Consensus**********************************************************************       860       870       880       890       900       910---------+---------+---------+---------+---------+---------| B29_promGCTCGGCCCTTCGGGGAGTT

GCAGAC

C

GAGG

CTGGCTGGCCCAGGG

T

B29_min Consensus************************************************************911    920       930       940       950       960       970       980|--------+---------+---------+---------+---------+---------+---------+B29_prom CCACCGGTGGGGTAAGCACAGACAG

GGGG

GCACAGGCTTCCCCCAGGA

ACTGAGAGGCCCCCCAGAG B29_min Consensus**********************************************************************       990      1000      1010      1020      1030      1040---------+---------+---------+---------+---------+---------| B29_promGCATCCACAGAGGACCCCAGCTGTGCTGCCCA

CTGG

GACC

CCTT

C B29_min                                       GCG

CC

CCTT

AC

C Consensus **************************************GC

CCTTA

1041  1050      1060      1070      1080      1090      1100      1110|--------+---------+---------+---------+---------+---------+---------+B29_prom

B29_min

Consensus

      1120      1130      1140      1150      1160      1170---------+---------+---------+---------+---------+---------| B29_prom

B29_min

Consensus

1171  1180      1190      1200      1210      1220      1230   1237|--------+---------+---------+---------+---------+---------+------|B29_prom

B29_min

Consensus

                                 C******************************

indicates data missing or illegible when filedStep 3: Subcloning of HA-eIF5A1^(K50R) into B29-5#3 Vector—CreatespB329-eIF5A1K50R_(—)7.

HA-eIF5A1^(K50R) was amplified by PCR using the pHM6-eIF5A1^(K50R) as aDNA template and the following primers: forward5′-CGCCATGGACATGTACCCTACGACGTCCCAGACTACGCTGCAGATGATTTG GACTTCGAG-3′ andreverse 5′-CGCGCTAGCAGTTATTTGCCATCGCC-3′. The resulting PCR fragment wasdigested with NcoI and NheI and subcloned into the NcoI and NheI sitesof B29-5 #3 to replace LacZ.

Sequence of HA-eIF5A1^(K50R) PCR fragment (497 bp) in pB29-eIF5A1K50R_7ACATGTACCCTTACGACGTCCCAGACTACGCTGCAGATGATTTGGACTTCGAGACAGGAGATGCAGGGGCCTCAGCCACCTTCCCAATGCAGTGCTCAGCATTACGTAAGAATGGTTTTGTGGTGCTCAAGGGCCGGCCATGTAAGATCGTCGAGATGTCTACTTCGAAGACTGGCAGGCATGGCCATGCCAAGGTCCATCTGGTTGGCATTGATATTTTTACTGGGAAGAAATATGAAGATATCTGCCCGTCGACTCATAACATGGATGTCCCCAACATCAAAAGGAATGATTTCCAGCTGATTGGCATCCAGGATGGGTACCTATCCCTGCTCCAGGACAGTGGGGAGGTACGAGAGGACCTTCGTCTGCCTGAGGGAGACCTTGGCAAGGAGATTGAGCAGAAGTATGACTGTGGAGAAGAGATCCTGATCACAGTGCTGTCCGCCATGACAGAGGAGGCAGCTGTTGCAATCAAGGCGATGGCAAAATAACTGTranslation of HA-eIF5A1^(K50R) PCR fragment in pB29-eIF5A1K50R_7HA epitope eIF5A1^(K50R) K50R MDMYPYDVPDYAADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCKIVEMSTSKTGRHGHAKVHLVGIDIFTGKKYEDICPSTHNMDVPNIKRNDFQLIGIQDGYLSLLQDSGEVREDLRLPEGDLGKEIEQKYDCGEEILITVLSAMTEEAAVAIKAM AKAlignment of HA-eIF5A1^(K50R) PCR fragment in pB29-eIF5A1K50R_7 with humaneIF5A1 (Accession # NP_001961)1       10        20        30        40        50        60        70|--------+---------+---------+---------+---------+---------+---------+eIF5A

HA-5A_K50R

Consensus ***********

       80        90       100       110       120       130--------+---------+---------+---------+---------+---------| eIF5A

HA-5A_K50R

Consensus

131    140       150       160  165 |--------+---------+---------+----+eIF5A

HA-5A_K50R

Consensus

indicates data missing or illegible when filedStep 4: Creation of New Multiple Cloning Site in pCpG-Mcs(Invivogen)—Creates pCpG-Linker4.

The pCpG cloning vector, pCpG-mcs G2 (Invivogen), was digested withEcoRI to remove the mammalian expression cassette containing the mCMVenhancer, the hEF1 promoter, the synthetic intron, the multiple cloningsite, and the SV40 polyadenylation signal. The EcoRI-digested pCpG-mcsG2 vector was then ligated to a synthetic linker with EcoRI sticky endsto create a promoterless vector with a new multiple cloning site(pCpG-Linker4).

Sequence of region surrounding new multiple cloning site in pCpG-Linker4

Step 5: Subcloning of B29 DS4.4 3′ Enhancer into pCpG-Linker—4-CreatespCpG-DHS4.4.

The B29 DHS4.4 3′ enhancer was amplified by PCR using pGEM-4.4enh #8 asa template and the following primers: forward5′-GAAGCGGCCGCACCACCCTGGGCCAGGCTGG-3′; and reverse5′-CCACGCGTAGAGGTGTTAAAAAGTCITTfAGGTAAAG-3′. The resulting PCR fragmentwas digested with NotI and MluI and ligated into the NotI and MluI sitesin the new multiple cloning site of pCpG-Linker4 to create pCpG-DHS4.4.

>pCpG-DHS4.4 full-length sequences (2,282 bp)

Step 6: Subcloning of B29 Promoter+HA-eIF5A1^(K50R)+SV40 pA ExpressionCassette into pCpG-DHS4.4-Creates pExp-5.

The B29-eIF5A1 expression cassette containing the minimal B29 promoter,the synthetic intron, HA-eIF5A1^(K50R), and the SV40 pA, was amplifiedfrom pB29-eIF5A1K50R_(—)7 (Step 3) by PCR using the following primers:forward 5′-GTTATCGATACTAGTGCGACCGCCAAACC-3′; and reverse5′-CAAGCGGCCGCCATACCACATTGTAGAGAGTTTTAC-3′. The resulting PCR fragmentwas digested with ClaI and NotI and subcloned into the ClaI and NotIsites in the multiple cloning site of pCpG-DHS4.4 to create pExp-5.

Step 7: Replacement of HA-eIF5A1^(K50R) in pExp-5 with non-HAeIF5A1^(K50R) Creates Final Vector, pExpSA.

The pExp-5 plasmid was digested with NcoI and NheI to removeHA-eIF5A1^(K50R). A non-HA-tagged eIF5A1^(K50R) PCR fragment wasamplified from pHM6-eIF5A1^(K50R) by PCR using the following primers:forward 5′-CACCATGGCAGATGATTTGGACTTC-3′; and reverse5′-CGCGCTAGCCAGTTATTTTGCCATCGCC-3′. The resulting PCR product wasdigested with NcoI and NheI and ligated into the NcoI and NheI sites ofB29-5 #3 to generate B29-K50R. B29-K50R was digested with NcoI and NheIand the 470 bp eIF5A1^(K50R) fragment was gel purified and ligated toNcoI/NheI-digested pExp-5 to generate the final expression vector,pExp5A.

Example 18 Testing of pExp5A

Various cell lines were transfected with plasmids using Lipofectamine2000 and expression of HA-eIF5A1K5OR was determined 24 hours followingtransfection by Western blotting with an anti-HA antibody (Roche).Different cells lines tested were P3X63Ag8.653 (mouse Blymphoblast—myeloma), KAS (hyman myeloma), HepG2 (huma liverhepatocellular carcinoma), T24 (human bladder carcinoma); HT-29 (humancolorectal adenocarcinoma), HEK-293 (human embryonic kidney cells), PC3(human prostrate adenocarcinoma); HeLa (human cervical adenocarcinoma),and A549 (lung carcinoma). See FIGS. 24A and 24B.

pExp-5 plasmid expresses HA-eIF5A1^(K50R) in both human and mousemyeloma cell lines at comparable levels to a plasmid with theconstitutive EF1 promoter (CpG-eIF5A1^(K50R)). However expression ofHA-eIF5A1^(K50R) driven by pExp-5 is limited in non-B cell linescompared to expression by a constitutive promoter. The one exception wasin HEK-293 cells, a human embryonic kidney cell line where high levelsof HA-eIF5A1^(K50R) expression was observed following pExp-5transfection—this may be due to 15 the embryonic nature of the cellline; at this time we do not know if pExp-5 expresses in adult kidneycells. The final plasmid for use in toxicity studies and clinical trialwill be a version of pExp-5 in which HA-eIF5A1^(K50R) has been replacedby non-HA tagged eIF5A1^(K50R) (pExp5A). pExp-5 contains HA-taggedeIF5A1^(K50R) under the control of the minimal human B29promoter/enhancer; expression of HA-eIF5A1^(K50R) was compared to thatdriven by plasmids with constitutive expression as well as to a plasmidcontaining the full-length B29 promoter

Example 19 Formation of In Vivo JETPEI™ Nanoparticle

This example given is for formation of the in vivo JETPEI™ nanoparticlecomplex for injection into 20 g mouse for a dose of 1.5 mg/kg (0.1mL)—1.5 mg/kg=1.0 mg pExp5A/kg+0.5 mg h5A1/kg—DNA:siRNA ratio=2:1.

Dilute plasmid DNA and siRNA into a total volume of 25 ml. Use sterilewater to adjust the volume. * Dilute 20 mg of plasmid DNA (10 ml at 2mg/ml) and 10 mg of siRNA (10 ml at 1 mg/ml) into a total volume of 25ml. Use sterile water to make up difference. Adjust the volume of DNAsolution to 50 ml 5% glucose by adding 25 ml of 10% glucose (providedwith PEI kit). Vortex gently and centrifuge briefly. Dilute in vivoJETPEI™ into a total volume of 25 ml of water. * Dilute 3.6 ml of invivo JETPEI™ into a total volume of 25 ml of water. Adjust final volumeto 50 ml with 10% glucose to end up with a final concentration of 5%glucose. Vortex gently and centrifuge briefly. Immediately add 50 ml ofdiluted PEI to the 50 ml of diluted DNA (do not reverse the order!).Vortex briefly and immediately spin down.

After formation the complex is stable for 8 to 10 hours. The N/P ratioof the complex should be 6. The N/P ratio is the ratio of the number ofpositively charged nitrogen residues of in vivo-jetPEI to the number ofnegatively charged phosphate residues of DNA and siRNA. DNA and siRNAcontain the same number of phosphate groups per gram. The N/P ratio istherefore a measure of the ionic balance within the complex. Increasingthe N/P ratio of the complex can increase the toxicity of the complex.In vivo JET-PEI is provided as a 150 mM solution (expressed as nitrogenresidues) while DNA contains 3 nmoles of anionic phosphate in 1 mg. Thefinal concentration of DNA in the final volume should not exceed 0.5mg/ml. The DNA should be of high quality and prepared in water. Invivo-jetPEI and 10% glucose should be brought to room temperature priorto use.

Example 20 Dose Range-Finding and Repeat Dose Studies with Intra-VenousSNS01 and SNS-EF1/UU in Mice

SNS01 is one embodiment of the present invention—it is a cancer therapybiologic targeted to the treatment of multiple myeloma. SNS01 iscomprised of three components: a DNA vector expressing a pro-apoptoticmutant of eIF5A (see FIG. 22); an siRNA that targets the native eIF5Athat promotes growth/anti-apoptosis of cancer cells (see the sequence inFIG. 25); and a synthetic polymer called polyethylenimine (Invivo-jetPEI; Polyplus Transfection Inc.) that acts as a deliveryvehicle.

The purpose of the studies was to determine the maximum tolerated doseand the feasibility of long-term administration of therapeutic doses ofintra-venous SNS01 into mice. Two separate studies were performed. Themaximum tolerated dose (Study ID: MTD) study was an 8-day study in whichmice received two intra-venous doses of increasing amounts of SNS01(from 2.2 mg/kg to 3.7 mg/kg) and toxicity was assessed by monitoringclinical signs, body weight, organ weight and liver enzymes. The 9-weekrepeated dose study (Study ID: EX6) was a study designed to assesstoxicity following long-term administration of twice-weekly therapeuticdoses (1.5 mg/kg) of SNS-EF1/UU and as well as it's various components.SNS-EF1/UU is a preclinical version of SNS01 and differs mainly in thatexpression of eIF5A^(K50R) is driven by a constitutive promoter (onethat expresses in all tissues at all times) rather than aB-cell-specific promoter as in the SNS01 complex. The use of the Bcell-specific B29 promoter in SNS01 was designed to enhance the safetyof the therapeutic by limiting expression of the pro-apoptotic eIF5Amutant to cells of B-cell origin, including myeloma cells. The EX6 studyalso included a group of mice that were dosed with a mouse-specificeIF5A siRNA to determine whether there were any toxic effects ofsuppressing eIF5A in mouse tissues. Toxicity in the repeated dose studywas assessed by monitoring clinical signs, body weight, hematology,liver enzymes, as well as histopathology.

Animal Injection Duration of Study ID Model Schedule Treatment TestArticle MTD CD-1 Twice weekly 8-Days SNS01 (female) Intra-venous (2injections) EX6 Balb/c Twice weekly 9-Weeks SNS-EF1/UU (female)Intra-venous SNS-EF1/UU components tested individually Mouse eIF5A siRNA

Test Article Plasmid DNA siRNA Vehicle Material Grade SNS01^(a)pExp5A^(c) eIF5A siRNA In vivo-jetPEI ™ GLP-grade (eIF5A^(K50R) (dTdToverhang^(d)) materials expression driven human-specific by B29 B-cell-eIF5A siRNA specific promoter) SNS-EF1/UU^(b) pCpG-HA- h5A1 Invivo-jetPEI ™ Research-grade eIF5A^(K50R) (UU overhang^(d)) materials(HA-eIF5A^(K50R) human-specific expression driven eIF5A siRNA ubiquitousEF1 promoter) ^(a)SNS01 contains GLP-grade materials and is beingdeveloped for the clinic ^(b)SNS-EF1/UU is a test article used inpreclinical research that led to the development of SNS01 ^(c)the pExp5Aplasmid is RNAi-resistant due to the fact that the plasmid contains onlythe open reading frame of eIF5A while the eIF5A siRNA (h5A1) targets the3′ UTR of eIF5A ^(d)the sequence of the SNS01 eIF5A siRNA and the h5A1siRNA are identical except for the presence of a dTdT overhang ratherthan a UU overhang on the 3′ terminal ends of the siRNA; the dTdToverhang does not affect the target selectivity or efficacy of the siRNAbut has been proposed to enhance stability

Preclinical experiments have indicated that SNS01 is therapeutic atdoses of 0.75 mg/kg to 1.5 mg/kg (Study EX9). In the 8-day doserange-finding study (Study ID: MTD) significantly higher doses than thetherapeutic range was tested to determine the upper limit of the doserange. Twice-weekly intra-venous doses of the test article was welltolerated at the lower dose levels of 2.2 mg/kg and 2.9 mg/kg althoughone mouse reached morbidity at the 2.9 mg/kg and was euthanized. Dosesat 3.3 mg/kg or above resulted in survival rates of approximately20-25%. Therefore, the maximum tolerated dose is between 2.2 mg/kg and2.9 mg/kg and is well above the therapeutic range of 0.75 mg/kg to 1.5mg/kg.

In the 9-week repeated dose study (Study ID: EX6) the mice receivedtwice weekly tail vein injections of therapeutic doses (1.5 mg/kg) ofSNS-EF1/UU and no test article-related toxic effects were observed overthe period of the study. The DNA and siRNA were also tested separatelyin this study and both were well tolerated by the mice. Since the humaneIF5A siRNA is not active in mice, a mouse eIF5A-specific siRNA was alsoincluded in this study. No toxic effects related to chronicadministration of the mouse eIF5A siRNA were observed over the 9-weekperiod. These results indicate that therapeutic doses of SNS01 andSNS-EF1/UU are non-toxic to mice even when administered over longperiods.

Test Article and Vehicle Manufacturer of Manufacturer of Plamid DNAsiRNA Manufacturer of Formation of Test Test Article component componentPEI component Article SNS01 VGXI Avecia Polyplus Components were Lot #Transfection Inc combined with pExp5A.08L007 GLP-Grade 10% glucose toform nano- complexes; complexes were injected within 2-4 hoursSNS-EF1/UU Qiagen EndoFree ThermoScientific Polyplus Components werePlasmid Mega Kit Dharmacon RNAi Transfection Inc, combined with <0.1EU/μg DNA Technologies Research-Grade 10% glucose to form nano-complexes; complexes were injected within 2-4 hours Storage −20° C. −20°4° Room Conditions (≧1 year) (≧1 year) (≧1 year) Temperature (Stability)(≧6 hours)

Test Systems and Study Designs

All aspects of this study were conducted in accordance with theguidelines set out by the University of Waterloo Animal Care Committee(Waterloo, Ontario, Canada) as established by the Canadian Council onAnimal Care and the Province of Ontario Animals for Research Act.The CD-1 and BALB/c mice were obtained from Charles River Laboratories(Quebec, Canada). Mice from both studies received the test articletwice-weekly via tail vein intra-venous injections. Slow injections(˜2-3 minutes) were used to deliver volumes greater than 0.2 ml.The mice for the 8-day study were approximately 6-9 weeks old at thestart of study. The mice for the 9-week repeated dose study wereapproximately 5-6 weeks old at the initiation of the study.

STUDY ID: MTD No. Dose Animals Test N/P level Injection Total # Group(female) Article ratio^(a) (mg/kg) Volume injections MTD-C 5 5% — — 0.33mL 2 Glucose MTD-PA 5 SNS01 6 2.2 mg/kg 0.20 mL 2 MTD-PB 5 SNS01 6 2.9mg/kg 0.27 mL 2 MTD-PC 4 SNS01 6 3.3 mg/kg 0.30 mL  1^(b) MTD-PD 5 SNS016 3.7 mg/kg 0.33 mL  1^(b) ^(a)N/P ratio = ratio of positively-chargednitrogens on PEI to the negatively-charged phosphate groups of thenucleic acids ^(b)due to toxicity the surviving mice were not given asecond injection

STUDY ID: EX6 No. Dose Animals Test N/P level Injection Total # Group(female) Article ratio (mg/kg) Volume injections Ex6-G1 4 5% — — 0.10 mL20 Glucose Ex6-G2 5 Vehicle 8 1.5 mg/kg 0.10 mL 20 Control^(a) Ex6-G3 5siRNA^(b) 8 1.5 mg/kg 0.10 mL 20 Ex6-G4 6 DNA^(c) 8 1.5 mg/kg 0.10 mL 20Ex6-G5 4 SNS-EF1/ 8 1.5 mg/kg 0.10 mL 20 UU Ex6-G6 6 Mouse 8 1.5 mg/kg0.10 mL 20 siRNA^(d) ^(a)PEI complex containing a non-expressing plasmid(same vector background as pExp5A) and a non-targeting siRNA ^(b)PEIcomplex containing a non-expressing plasmid (same vector background aspExp5A) and tne h5A1 siRNA ^(c)PEI complex containingpCpG-HA-eIF5A^(K50R) plasmid and a non-targeting siRNA ^(d)PEI complexcontaining a non-expressing plasmid (same vector background as pExp5A)and a mouse-specific eIF5A siRNA (the human eIF5A siRNA is not active inmouse)

8-Day Maximum Tolerated Dose Study (MTD)

The two-dose 8-day study was a dose range-finding study designed todetermine the maximum tolerated dose of SNS01. The dose range was 2.2mg/kg to 3.7 mg/kg and is well above the therapeutic dose range of 0.75mg/kg to 1.5 mg/kg. At the lowest dose (2.2 mg/kg) of SNS01 no clinicalsigns of toxicity were observed except for one mouse that exhibitedslightly ruffled fur and decreased activity that resolved within onehour. No clinical signs of toxicity were observed following the 2^(nd)injection of 2.2 mg/kg of SNS01. All the mice maintained their weightthroughout the study. No macroscopic changes in the organs wereobserved. The organ weight to body weight ratios were unchanged from thecontrol group except for a modest increase in the ratio of the liverweight:body weight ratio. However, since an increase in this ratio wasnot observed in any of the higher dose level groups it is unlikely to berelated to the test article.

Four out of five mice tolerated 2.9 mg/kg of SNS01 with no clinicalsigns of toxicity. However, one mouse experienced convulsions and mildrespiratory distress within 1 hour of injection and had to be humanelyeuthanized. No clinical signs of toxicity were observed following the2^(nd) injection of SNS01 in the remaining mice. The mice maintainedtheir weight throughout the study and no macroscopic changes in theorgans or changes in the organ weight to body weight ratios wereobserved. There was a slight increase in the serum levels of ALTfollowing two doses of 2.9 mg/kg SNS01.

As expected, doses of SNS01 at or above 3.3 mg/kg were not welltolerated and in both groups all mice but one had to be humanelyeuthanized. In all cases where mice were humanely euthanized due tomorbidity the clinical signs appeared within 1 hour of injection andwere consistent with other reported studies using high doses of PEI. Thesurviving mice of the 3.3 mg/kg and 3.7 mg/kg recovered completelywithin 4 hours after the injection and maintained their weightthroughout the study, although they did not receive a 2^(nd) dose. Themaximum tolerated dose of SNS01 therefore appears to be between 2.2mg/kg and 2.9 mg/kg.

9-Week Repeated Dose Study (EX6)

The purpose of the 9-week repeated dose study was to assess the safetyof chronic administration of therapeutic doses (1.5 mg/kg) ofSNS-EF1/UU, a complex that was used for preclinical studies duringdevelopment of SNS01. SNS-EF1/UU does not differ significantly fromSNS01, the major difference being that the materials are research-gradeand that eIF5A^(K50R) expression is driven by the constitutive human EF1promoter that is active in all cell types. Although SNS01 uses aB-cell-specific promoter to drive eIF5A^(K50R) expression, the use of aconstitutive promoter in this safety study allows for the assessment oftoxicity resulting from the accumulation of the mutant eIF5A^(K50R)protein in non-B-cell tissues. Another aspect of the 9-week repeateddose study was the inclusion of groups to test the safety of theindividual components of SNS-EF1/UU. The DNA group (Ex6-G3) was dosedwith a complex containing the eIF5A plasmid and a non-targeting controlsiRNA while the siRNA group (Ex6-G4) was dosed with a complex containingthe human eIF5A (h5A1) siRNA and a non-expressing plasmid. Since thetest article SNS-EF1/UU contains a human eIF5A siRNA that will notaffect expression of the endogenous mouse eIF5A. another feature of thisstudy was the inclusion of a group (Ex6-G6) that was dosed with PEIcomplexes containing a non-expressing plasmid and an siRNA thatefficiently targets mouse eIF5A. This group allowed assessment of thesafety of chronic administration of an active eIF5A siRNA.

All animals survived to the scheduled sacrifice date. No clinical signsof toxicity were observed in any of the groups over the course of the9-week study and the mice in all groups continued to gain weight duringthe study period. Red and white blood cell counts were measured threeand six weeks after the initiation of treatment and were normal for alldosing groups. Serum liver enzyme levels were measured followingsacrifice and were within the normal range for all mice. No changes inthe macroscopic appearance of the organs were observed in any of thegroups. Histopathological analysis of the major organs was conducted byan independent pathologist (and revealed no toxicity attributable to thetest article.

Chronic administration of therapeutic doses of SNS-EF1/UU is welltolerated by mice and no adverse effects were observed. In addition,chronic administration of a mouse-specific eIF5A siRNA revealed no toxiceffects, indicating that the administration of PEI complexes containinga human eIF5A siRNA should be safe for humans.

Example 21 Therapeutic Efficacy Studies with Intra-Venous SNS-B29/UU andSNS01 in Multiple Myeloma Tumour-Bearing Mice

SNS01 is as described above. The test article SNS-B29/UU is apreclinical version of SNS01. SNS-B29/UU differs very little from SNS01,the chief difference being that the components are of research-graderather than GLP-grade. The purpose of the study reported here was todetermine the minimum effective dose of SNS-B29/UU and to confirm thatthe GLP-grade materials that comprise SNS01 perform as well as theresearch-grade materials that were used for the preclinical studies. Therepeated dose tumour study (Study ID: EX9) was a 5-week study in whichthe ability of increasingly smaller twice-weekly doses of SNS-B29/UU toinhibit subcutaneous tumour growth in mice was assessed in order todetermine the optimal therapeutic dose of SNS01. The treated animalswere also assessed for signs of toxicity by monitoring clinical signs,body weight and organ weight.

Injection Duration of Study ID Animal Model Schedule Treatment TestArticle Ex9 C.B17 (SCID) Twice weekly 35 Days SNS-B29/UU (female) miceIntra-venous bearing Twice weekly 25 Days SNS01 subcutaneousIntra-venous human multiple myeloma tumours (KAS-6/1)

Test Article Plasmid DNA siRNA Vehicle Material Grade SNS01^(a)pExp5A^(c) eIF5A siRNA^(d) In vivo-jetPEI ™ GLP-grade (eIF5A^(K50R)(dTdT overhang^(c)) materials expression driven human-specific by B29B-cell- eIF5A siRNA specific promoter) SNS-B29/UU^(b) pExp5A^(c) h5A1 Invivo-jetPEI ™ Research-grade (UU overhang^(c)) materials human-specificeIF5A siRNA ^(a)SNS01 contains GLP-grade materials and is beingdeveloped for the clinic ^(b)SNS-B29/UU is a test article used inpreclinical research that led to the development of SNS01 ^(c)the pExp5Aplasmid is RNAi-resistant due to the fact that the plasmid contains onlythe open reading frame of eIF5A while the eIF5A siRNA (h5A1) targets the3′ UTR of eIF5A ^(d)the sequence of the eIF5A siRNA and the h5A1 siRNAare identical except for the presence of a dTdT overhang rather than aUU overhang on the 3′ terminal ends of the siRNA; the dTdT overhang doesnot affect the target selectivity or efficacy of the siRNA but has beenproposed to enhance stability

The therapeutic range of SNS-B29/UU was determined in SCID mice bearingsubcutaneous human multiple myeloma tumours. Doses of SNS-B29/UU between0.15 mg/kg and 1.5 mg/kg were tested. The anti-tumoural efficacy of thetest article was determined by twice-weekly tumour volume measurementsand by excising and weighing tumour tissue following sacrifice.SNS-B29/UU doses of 0.75 mg/kg and 1.5 mg/kg resulted in significanttumour shrinkage indicating that the therapeutic range of SNS-B29/UUlies between 0.75 mg/kg and 1.5 mg/kg. Effective inhibition of growth ofsubcutaneous tumours was also observed at 0.38 mg/kg SNS-B29/UU althoughno tumour shrinkage was observed. Some inhibition of tumour growth waseven observed at doses as low as 0.15 mg/kg SNS-B29/UU indicating abroad therapeutic range. See FIGS. 26 and 27.

The efficacy of SNS01 made using GLP-grade components was compared toSNS-B29/UU and was found have a comparable efficacy in the inhibition oftumour growth. Treatment of tumour-bearing SCID mice with SNS01 andSNS-B29UU was well tolerated and the mice continued to gain weightthroughout the study.

Test Article and Vehicle Manufacturer of Manufacturer of Plasmid DNAsiRNA Manufacturer of Formation of Test Test Article component componentPEI component Article SNS01 VGXI Avecia Polyplus Components were Lot #Transfection Inc combined with pExp5A.08L0007 GLP-Grade 10% glucose toform nano- complexes; complexes were injected within 2-4 hoursSNS-B29/UU Qiagen EndoFree ThermoScientific Polyplus Components werePlasmid Mega Kit Dharmacon RNAi Transfection Inc. combined with <0.1EU/μg DNA Technologies Research-Grade 10% glucose to form nano-complexes; complexes were injected within 2-4 hours Storage −20° C. −20°4° Room Conditions (≧1 year) (≧1 year) (≧1 year) Temperature (Stability)(≧6 hours)

Test Systems and Study Design

The female C.B.17/IcrHsd-Prkdc (SCID) mice were obtained from Harlan(Indianapolis, Ind., USA). Subcutaneous tumours were established byinjecting 10×10⁶ viable KAS-6/1 (human multiple myeloma) cells into theright flank of 5 to 6 week-old mice. Treatment with SNS-B29/UU beganwhen the tumours reached an approximate size of 20 to 40 mm³(approximately 4 weeks after tumour cell injection). Treatment withSNS01 began when the tumours reached an approximate size of 130 mm³(approximately 6 weeks after tumour cell injection). Mice received thetest article twice-weekly via tail vein intra-venous injection.

STUDY ID: EX9 No. Dose Total # Animals Test N/P level Injection injec-Group (female) Article ratio (mg/kg) Volume tions Ex9-G1 3 Control^(a) 6 1.5 mg/kg 0.1 mL 11 Ex9-G2 4 SNS- 6  1.5 mg/kg 0.1 mL 11 B29/UU Ex9-G34 SNS- 6 0.75 mg/kg 0.05 mL  11 B29/UU Ex9-G4 4 SNS- 6 0.38 mg/kg 0.025mL  11 B29/UU Ex9-G5 3 SNS- 6 0.15 mg/kg 0.01 mL  11 B29/UU Ex9-G9 3SNS01 6  1.5 mg/kg 0.1 mL 7 ^(a)PEI complex containing a non-expressingplasmid (same vector background as pExp5A) and a non-targeting siRNA

Repeated Dose Tumour Study

The repeated dose tumour study was designed to determine the minimumeffective therapeutic dose of SNS-B29/UU and to confirm that theGLP-grade SNS01 test article retained the tumour inhibition activitydemonstrated by the research-grade test article SNS-B29/UU. A secondaryobjective was to assess any toxic effects of the treatment by monitoringthe treated mice for clinical signs, body weight, and organ weight. Testarticle therapeutic anti-tumoural activity was monitored by twice-weeklytumour volume measurements using digital calipers. Upon sacrifice thetumours were excised and weighed.

All the mice survived to the scheduled sacrifice date. Control mice thatwere treated with PEI nanocomplexes containing a non-expressing plasmidand a non-targeting siRNA had an average tumour volume of 284 mm³ at thetime of sacrifice while mice treated with 1.5 mg/kg SNS-B29/UU had anaverage tumour volume of only 13 mm³, a 95% (*p=0.026) reduction intumour growth. However, when it was attempted to excise the tumours frommice that had been treated with 1.5 mg/kg SNS-B29/UU, no evidence of atumour was found in any of the mice. Decreasing the dose of SNS-B29/UUby half to 0.75 mg/kg still resulted in a 91% (*p=0.03) and 87%(*p=0.04) decrease in tumour volume and weight, respectively, and in onemouse the tumour had completely disappeared. Therefore, the optimumtherapeutic dose for twice-weekly injections of SNS-B29/UU appears to bebetween 0.75 mg/kg and 1.5 mg/kg. Twice-weekly doses of SNS-B29/UU dosesas low as 0.15 mg/kg still resulted in a 60% reduction in the finaltumour volume indicating that SNS-B29/UU has potent anti-tumouralactivity over a wide dose range.

In addition to inhibiting tumour growth, treatment with SNS-B29/UU andSNS01 at 0.75 mg/kg and 1.5 mg/kg resulted in significant reduction intumour volume indicating that this treatment is capable of inducingtumour regression, likely through the induction of apoptosis in thetumour. The percent change in tumour volume in tumour-bearing micetreated with SNS-B29/UU, at dose levels of 0.75 mg/kg and 1.5 mg/kg, was−244% and −245%, respectively. The tumours of control mice increased insize by more than 2000% during the same time period. Twice-weeklyinjections of SNS01 also significantly shrunk multiple myeloma tumours.The percent change in tumour volume for mice treated with 1.5 mg/kgSNS01 was −349%, indicating that SNS01 is just as effective asSNS-B29/UU. The use of GLP-grade materials may in fact have increasedthe biological activity since treatment with SNS01 achieved a 349%decrease in tumour volume following only 25 days of treatment whileSNS-B29/UU achieved a 245% reduction in tumour volume following 35 daysof treatment. In addition, the tumours treated with SNS01 were quitelarge (˜130 mm³) indicating that treatment with SNS01 is effectiveagainst well-established tumours.

The treatment was well tolerated by all mice and no clinical signs oftoxicity were observed. The mice in all groups continued to gain weightthroughout the study. No changes in the macroscopic appearance of theorgans was observed at necropsy and no significant changes in the organweight to body weight ratios occurred.

Therefore SNS01 (and its preclinical version SNS-B29/UU) is welltolerated by SCID mice and is extremely effective in treatingsubcutaneous human multiple myeloma tumours when delivered byintra-venous injection twice per week. All doses of SNS-B29/UU that weretested were effective at inhibiting tumour growth but the highest doseof 1.5 mg/kg successfully eliminated tumours in all mice receivingtreatment.

Example 22 Biodistribution of Plasmid DNA and siRNA Polyethylenimine(JetPEI) Complexes

Green fluorescent protein (“GFP”) GFP-expression constructs were used todetermine localization of plasmid DNA delivered by PEI complexes. Twopromoters were used to drive GFP expression: EF1: ubiquitous promoter(EF1::GFP) or B29: B-cell specific promoter (B29::GFP). PEI complexescontaining 20 micrograms of GFP plasmid DNA and 10 micrograms of afluorescently-labelled (DY547) h5A1 siRNA were prepared at an N/P ratioof 6. BalB/C mice were injected intra-venously with either 5% glucose orPEI complexes on two consecutive days. Seventy-two hours following thefirsts injection the mice were euthanized and their organs wereharvested and analyzed for GFP expression and DY547-siRNA by confocalmicroscopy.

Bone Marrow:

In most cases there was evidence of DY547-siRNA but no GFP expression.Timing of organ harvest may not coincide with peak expression of GFP;and there may be quenching of GFP signal or GFP may not be expressed.However, GFP and DY547 that colocalized to the same bone marrow cells insome instances was observed. Therefore, this provides evidence that PEInanoparticles can transfect bone marrow cells in a live animal whengiven by intra-venous injection

Lung:

In most cases there was evidence of DY547-siRNA but no GFP expression.Timing of organ harvest may not coincide with peak expression of GFP orthere may be quenching of GFP signal or GFP may not be expressed.

Spleen:

Evidence of GFP expression (when driven by EF1 promoter) colocalizing incells also positive for the presence of DY547-siRNA was seen. Expressionof GFP was much lower in spleen cells when driven by the B29 promoter.This shows that PEI nanoparticles appear to transfect cells of thespleen.

Kidney:

No GFP or DY547 was observed indicating nanoparticles may not enterkidney.

Liver:

In most cases there was evidence of DY547-siRNA but no GFP expression.This provides evidence that PEI nanoparticles are transfecting cells ofthe liver.

Heart:

Colocalization of EF1::GFP and DY547-siRNA in tissue of heart was seen,thus indicating that PEI nanoparticles may be transfecting this organ.No GFP was observed with B29 promoter.

Example 23 Effect of DNA:siRNA Ratio on HA-eIF5A^(K50R) Expression

KAS cells were transfected with nanoparticles containingB29-HA-eIF5A^(K50R) (plasmid driven by B-cell-specific promoter) andh5A1 siRNA. JET PEI™ nanoparticles containing different ratios of pExp5Aand h5A1 siRNA were made and incubated for 4 hours at room temperatureprior to addition to KAS cells. Four hours after transfection, thenanoparticle-containing media was replaced with fresh media. Twenty-fourhours later the cell lysate was harvested and used for western blotanalysis with an antibody against HA. The ratio of DNA:siRNA was variedfrom the standard ratio of 2:1. The accumulation of HA-eIF5A^(K50R)peaked at ratios of 1:0, 3:1, and 2:1. See FIG. 30.

Example 24 Effect of DNA:siRNA Ratio on Apoptosis Induced byNanoparticle Transfection

Nanoparticles containing different ratios of pExp5A and h5A1 siRNA weremade and incubated for 4 hours at room temperature prior to addition toKAS cells. Four hours after transfection, the nanoparticle-containingmedia was replaced with fresh media. Forty-eight hours later the cellswere harvested, labelled with Annexin V/PI and analyzed by FACS. Theinduction of apoptosis was highest in cells transfected withnanoparticles with the standard DNA:siRNA ratio of 2:1. See FIG. 31.

Example 25 Administration of PEI Complexes (N/P=6 or 8) ContainingeIF5A1K50R Plasmid and eIF5A1 siRNA (siSTABLE or Non-siSTABLE) InhibitsGrowth of Multiple Myeloma Subcutaneous Tumours and Results in TumourShrinkage

SCID mice were injected subcutaneously with KAS cells. Treatment wasinitiated when palpable tumours were observed. Mice were injectedintra-venously 2 times per week with either: (G1) PEI complexescontaining 20 mg of pCpG-mcs (empty vector) and 10 mg of control siRNAat N/P=8 (medium dose); (G5) PEI complexes containing 20 mg of theRNAi-resistant plasmid pCpG-eIF5A1k50R and 10 mg of siSTABLE h5A 1 siRNAat N/P=8 (Imedium dose, siSTABLE); (G8) PEI complexes containing 20 mgof the RNAi-resistant plasmid pCpG-eIF5A1k50R and 10 mg of h5A1 siRNA atN/P=6 (medium dose, N/P=6). The data shown is the individual tumourvolume for the mice in each group. The final injection was given at day40 after initiation of treatment. See FIG. 32.

Example 26 JET PEI™ Nanoparticles are being Effectively Taken Up byTumour Tissue and that Nanoparticles are Delivering Plasmid and siRNA tothe Same Cell

Tumour section 48 hours after injection with nanoparticles containingpExp-GFP (GFP under control of B-cell-specific promoter) and DY547-siRNA(fluorescently-labelled siRNA). Co-localized expression of GFP and DY547is observed in tumour section following confocal microscopy indicatingthat the nanoparticles are being effectively taken up by tumour tissueand that nanoparticles are delivering plasmid and siRNA to the samecell. See FIG. 33.

1. A composition comprising a complex of an eIF5A1 siRNA targetedagainst the 3′ end of eIF5A1, an expression vector comprising apolynucleotide encoding a mutant eIF5A1 wherein the mutant eIF5A1 isunable to be hypusinated, and wherein the siRNA and the expressionvector are complexed to polyethylenimine to form a complex.
 2. Acomposition comprising an siRNA targeted against a target gene tosuppress endogenous expression of the target gene in a subject; and apolynucleotide encoding a target protein capable of being expressed inthe subject in an RNAI resistant plasmid, wherein the siRNA and theplasmid are complexed to polyethylenimine to form a complex.
 3. Thecomposition of claim 1 wherein the siRNA has the sequence shown in FIG.25 and wherein the polynucleotide encoding the mutant eIF5A1 iseIF5A1^(K50R).
 4. The composition of claim 3 comprising a tissuespecific promoter.
 5. The composition of claim 4 comprising a B cellspecific promoter.
 6. The composition of claim 5 wherein the B cellpromoter is B29.
 7. The composition of claim 3 wherein the expressionvector comprises a pCpG plasmid.
 8. The composition of claim 1 whereinthe eIF5A1 siRNA and the expression vector comprising the mutant eIF5A1polynucleotide are independently complexed to polyethylenimine.
 9. Thecomposition of claim 1 wherein the eIF5A1 siRNA and the expressionvector comprising the mutant eIF5A1 polynucleotide are togethercomplexed to polyethylenimine.
 10. A composition comprising an eIF5A1siRNA targeted against the 3′ end of eIF5A1 and an expression vectorcomprising a polynucleotide encoding a mutant eIF5A1 wherein the mutanteIF5A1 is unable to be hypusinated, and wherein the siRNA and theexpression vector are delivered to a subject to treat cancer.
 11. Thecomposition of claim 10, wherein the cancer is multiple myeloma.
 12. Amethod of treating cancer comprising administering the composition ofclaim 10 to a subject.
 13. A method of treating cancer comprisingadministering the composition of claim 1 to a subject.
 14. The method ofclaim 12 wherein the composition is administered intravenously, intraperitoneally or intra tumorally.
 15. The method of claim 13 wherein thesiRNA targeted against the 3′ end of eIF5A1 and the expression vectorcomprising the polynucleotide encoding a mutant eIF5A1 are delivered viadifferent routes.
 16. The method of claim 12 wherein the composition isprovided at a dose of about 0.15 mg/kg to about 1.5 mg/kg for twiceweekly injections.
 17. The method of claim 12 wherein the composition isprovided at a dose of about 0.75 mg/kg to about 1.5 mg/kg for twiceweekly injections.